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Cellular structure and function 

Cellular structure and function
Cellular structure and function
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date: 27 October 2021

General principles: overview

There are four major chemical components to biological life—carbon (C), hydrogen (H), oxygen (O), and nitrogen (N):

  • O, C, and H form the bulk of the dry mass—65%, 18%, and 9% respectively of the human body (e.g. carbohydrates, simple lipids, hydrocarbons)

  • N represents 4% and is an essential part of life (nucleotides, amino acids, amino sugars, complex lipids)

  • They are supplemented by phosphorus (P) in the form of phosphate (PO43–) and by small amounts of sulphur (S) in the amino acids methionine and cysteine, and in thioester bonds

  • There are also a number of essential trace elements (e.g. Mn, Zn, Co, Cu, I, Cr, Se, Mo) that are essential co-factors for enzymes.

Note that all biological molecules (biomolecules) must obey the basic rules of chemistry!

One important such example for C atoms is stereochemistry:

  • Three-dimensional (3D) structures in biomolecules are often key to their function

  • Carbon atoms can have four different groups attached to them (i.e. be tetrahedral)

  • Under these conditions, the C atom is said to be chiral—it has two mirror-image forms (d- and l-) that cannot be superimposed (Fig. 1.1)

  • Nature has favoured certain stereoisomers:

    • For example, naturally occurring mammalian amino acids are always in the l-form, whereas carbohydrates are always in the d-form

    • The stereoisomers of therapeutic compounds may have very different effects:

      • For example, one stereoisomer of the drug thalidomide (a racemic mix), used briefly in the late 1950s to relieve symptoms of morning sickness in pregnancy, caused developmental defects in ~12,000 babies.

Fig. 1.1 Stereoisomers: the molecule on the left cannot be superimposed on the one on the right however it is rotated.

Fig. 1.1 Stereoisomers: the molecule on the left cannot be superimposed on the one on the right however it is rotated.


Roughly speaking, biomolecules can be divided up into two groups: small and large (macromolecules).

  • Small—relative molecular mass (Mr) <1000 (mostly <400):

    • Intermediates of metabolic pathways (metabolites), and/or

    • Components of larger molecules

  • Macromolecules—Mr >1000, up to millions:

    • Usually comprised of small biomolecule building blocks, for example:

      • Proteins (made up of amino acids)

      • Polysaccharides (sugars)

      • DNA, RNA (nucleotides, themselves sugars, bases, and phosphate)

    • Macromolecules can contain more than one type of building block, e.g. glycoproteins (amino acids and sugars).

How are larger biomolecules formed from small ones?

When smaller molecules are to be combined into larger ones, often the smaller molecules are activated in some way before the joining reaction takes place. This is because firstly, often the activation will allow an otherwise energetically unfavourable reaction to take place and, secondly, it may well influence the reaction that takes place to ensure that the correct product is formed.

What roles do macromolecules play in cell structure and function?

There are four basic types of macromolecule in cells:

  • Proteins—probably have one of the broadest range of functions in the body of any of the macromolecules. For example:

    • Enzymes (biological catalysts)

    • Structural

    • Membrane transporters (channels, carriers, pumps)

    • Receptors

    • Signalling molecules, e.g. hormones

  • Lipids—lipid molecules do not polymerize, but can associate non-covalently in large numbers:

    • Major component of cell membranes (plasma and organelle)

    • Energy storage

    • Signalling (intracellular, hormone)

    • Insulation (electrical in nerves, thermal)

    • Components of other macromolecules (e.g. lipoproteins, glycolipids)

  • Carbohydrates (polysaccharides)—also have a diverse range of functions in the body, including:

    • Structural (connective tissue)

    • Cell surface receptors (short chains linked to lipids and proteins)

    • Energy storage (glycogen)

    • Source of building blocks for other molecules (e.g. conversion to fat, amino acids, nucleic acids)

  • Nucleic acids (DNA/RNA)—transfer of genetic information from generation to generation (DNA) and for determining the order of amino acids in proteins (RNA).

Proteins: overview

Proteins have a wide range of biological functions, determined ultimately by their 3D structures (in turn determined by primary sequence) and potentially by higher levels of organization (i.e. multi-subunit complexes).

Proteins fall into a number of functional types.


  • Can be extracellular (‘fibrous’), e.g. in bone (collagen), skin, hair (keratin):

    • May use disulphide bonds to stabilize structure → strength

  • Intracellular structural proteins, e.g. cytoskeletal proteins such as actin, fibrin (blood clots).


Describes most non-structural proteins:

  • Generally compact due to folding of polypeptide chain

  • Usually bind other molecules (ligands), e.g. haemoglobin and O2, antibody and epitope, ferritin and iron:

    • Binding is specific and involves a particular part of the protein (binding site):

      • Can be tight (high affinity) or loose (low affinity) or anywhere in between

    • Ligand binding can alter the function of the protein.

Enzymes are a specific type of globular protein:

  • Bind substrate(s) and catalyse conversion into product(s) (reactants)

  • Enzymes are catalysts, i.e. are chemically identical before and after the reaction they catalyse

  • Enzymes do not change reaction equilibrium, but increase the rate at which equilibrium is reached:

    • This is achieved by lowering the reaction activation energy.

Plasma membrane proteins

  • Integral membrane proteins, e.g. transporters, receptors:

    • Generally, compact globular proteins

    • Have hydrophobic domains in the membrane lipid and hydrophilic domains in the cytoplasm/extracellular fluid

  • Membrane-anchored proteins:

    • Attached by covalent bonds to membrane elements, e.g. phospholipid head group.

Regulatory proteins

These are proteins which affect the activity of other proteins, for example:

  • Protein kinases (PKs), which phosphorylate proteins and affect their function, e.g. PKA, PKC, tyrosine kinase

  • Calmodulin, a Ca2+ binding protein, can be part of multi-subunit enzyme complexes and regulate their activity.

Amino acids and the peptide bond

Amino acids are the building blocks from which proteins are formed, by the joining of these simple subunits into polymers (Table 1.1).

Table 1.1 Amino acids


3-letter code

Single-letter code

Side group (except for proline where whole amino acid is shown)












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–CH2 – OH




–CH2 – SH




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–CH2 – CH2 – S – CH3




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–CH2 – CH2 – CH2 – CH2 – NH+3




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All amino acids have the same basic structure (Fig. 1.2).

Fig. 1.2 Basic structure of amino acids; R is the side-chain.

Fig. 1.2 Basic structure of amino acids; R is the side-chain.

There are 20 common side-chains (R) in the amino acids that are found in mammalian proteins, and they fall into five broad categories (NB: amino acids can be in more than one category; Fig. 1.3):

  1. 1. Hydrophilic, e.g. serine, glutamine

  2. 2. Hydrophobic, e.g. phenylalanine, valine

  3. 3. Basic—arginine, lysine

  4. 4. Acidic—aspartate, glutamate

  5. 5. ‘Structural’, e.g. proline.

Fig. 1.3 Amino acids grouped by physicochemical characteristics (see Table 1.1 for the single letter amino acid codes).

Fig. 1.3 Amino acids grouped by physicochemical characteristics (see Table 1.1 for the single letter amino acid codes).

  • Cysteine and methionine contain sulphur

  • The amino group of the side-chain of arginine and lysine has a pK of ~14, so is always positively charged under physiological conditions

  • The carboxyl group of the side-chain of aspartate and glutamate has a pK of ~4, so is always negatively charged under physiological conditions

  • Histidine side-chain has a pK of ~6 so can be either protonated (basic) or unprotonated (neutral) under physiological conditions

  • Proline is known as a ‘structural’ amino acid as it puts a kink in polypeptide chains:

    • Glycine also allows flexibility in polypeptide chains due to the small size of its R group (i.e. a proton).

All the amino acids in mammalian proteins are the l stereoisomer.

  • Bacteria make use of d-isomers:

    • Some antibiotics mimic d-amino acid and thus interfere with bacterial metabolism.

Amino acids are joined together in polypeptides by peptide bonds.

  • Methionine is the starting amino acid of all proteins

  • The peptide bond (–CO–NH–) is formed by a condensation reaction

  • The peptide bond is planar and rigid (trans configuration), with no rotation around C–N bond

  • Rotation around Cα‎–C and N–Cα‎ bonds allows polypeptide chains to form 3D shape of proteins.

Protein function is determined from the primary amino acid sequence.

  • The order of the amino acids will give the protein its shape

  • The 3D shape of the protein is essential to its function, e.g. the shape of the substrate binding site of an enzyme—this includes the position of the R groups, which is determined by the stereoisomer (l vs d).

Principles of protein structure

Proteins are held together at a molecular level by a number of forces:

  • Van der Waals’:

    • The weakest interactions, also known as London forces

  • Hydrogen bonding:

    • Non-covalent; occurs between electronegative atoms (N or O) and an electropositive proton, e.g. =N … NH=, =N … HO, C=O … HN=

  • Hydrophobic forces:

    • Interactions between groups of hydrophobic amino acids to reduce the amount of water associated with them and thus to increase thermodynamic stability of structure

    • Strong driving force for globular protein formation

    • Can also occur between hydrophilic amino acids in non-aqueous surroundings

  • Ionic interactions:

    • Between R groups of oppositely charged amino acids, e.g. arginine—glutamate, aspartate—lysine

  • Disulphide bonds:

    • The only covalent bonding involved in protein structure

    • Occur between the –SH groups of cysteines, to give –S–S– bonds. These bonds can be broken by denaturing the protein, e.g. with heat or solvents. Denaturation can be reversible or irreversible.

Proteins have four levels of organization

  1. 1. Primary—the order of the amino acids, e.g. Gly–Ala–Val.

  2. 2. Secondary—the formation of regions of structure in the polypeptide chain. These are stabilized by hydrogen bonds between the C=O and N–H moieties of peptide bonds in the polypeptide chain. Examples of such regions of local structure within the protein are the:

    • α‎-helix (Fig. 1.4):

      • Most stable of several possible helical structures which amino acids can adopt due to least strain on the inter-amino acid hydrogen bonding

      • R groups point outwards from the helix. Usual form protein adopts when crossing lipid bilayers

      • Stabilized by hydrogen bonds between every fourth peptide bond; the peptide bonds are adjacent in 3D space (periodicity of helix = 3.6 residues)

    • β‎-sheet (Fig. 1.4):

      • Polypeptide chain is in a fully extended conformation

      • Can run parallel or anti-parallel

      • Stabilized by hydrogen bonds between peptide bonds of adjacent polypeptide chains

    • Loop/turn or β‎-turn (Fig. 1.4):

      • Region where polypeptide chain makes a 180° change in direction, often at surface of globular proteins

      • β‎-turns are stabilized by hydrogen bonds between residues 1 and 4 of this four-amino acid structure. Residues 2 and 3 are at the end of the loop and are often Pro or Gly, and do not contribute stabilizing bonds

      • Loops can have a more complex structure.

  3. 3. Tertiary—the overall 3D structure formed by a polypeptide chain.

    • Describes the overall arrangement of the regions of secondary protein structure:

      • Recognizable combinations of secondary structures (super secondary structures, or motifs) may be present in different proteins, e.g. α‎-helix–turn–α‎-helix is often a DNA binding motif

      • Certain more stand-alone structural regions (domains) may be identified whose function is known, e.g. an ATP binding domain

      • Families of functionally related proteins may have a similar tertiary structure. For example, ATP-binding cassette (ABC) proteins have two domains, each of six membrane-spanning (α‎ helical) regions and an ATP-binding site.

    • Stabilized by the forces described earlier (van der Waals’, hydrogen bonding, hydrophobic forces, ionic interactions and potentially disulphide bonds) including between the R groups

    • The primary structure, i.e. the amino acid sequence, determines the tertiary (3D) structure of a protein. This was shown by Christian Anfinsen in 1961 when the enzyme ribonuclease spontaneously refolded into its active native structure after chemical denaturation1

    • Proteins fold in an at least partially defined pathway with intermediates between the unfolded and dully folded forms—they fold far too quickly to try all possible conformations

    • In aqueous globular proteins, one of major determinants of 3D structure is the packing of the hydrophobic residues in the interior with water excluded (hydrophobic forces):

      • The C=O and NH groups of the peptide bonds are all hydrogen bonded to each other in α‎-helices and β‎-sheets, and van der Waals’ bonds stabilize the hydrocarbon backbones

      • The outer surface has the hydrophilic residues, including those found in the turns/loops

      • The amino acid sequence often leads to amphipathic structures, such as α‎-helices and β‎-sheets where one side is hydrophilic and one side hydrophobic, allowing these to interact with two environments

      • Proteins in hydrophobic environments, such as membrane proteins, need to have the opposite orientation.

  4. 4. Quaternary—the interaction of a number of polypeptide chains to form a multimeric protein complex.

    • Many proteins are made up of more than one subunit, with the two or more polypeptide chains held together usually by the weak non-covalent interactions previously described:

      • The resulting oligomers can be either homo-oligomers (made up of a number of identical subunits) or hetero-oligomers (made up of different subunits)

    • Misfolding of proteins is associated with some neurological diseases, e.g. Parkinson’s disease, Huntington’s disease, and Creutzfeldt–Jakob disease (CJD):

      • Misfolded forms of normally soluble proteins accumulate as amyloid fibrils or plaques (hence the diseases are collectively known as amyloidoses)

      • The insoluble amyloid fibrils are rich in β‎-sheets.

Fig. 1.4 Diagram of the structure of staphylococcal nuclease protein.

Fig. 1.4 Diagram of the structure of staphylococcal nuclease protein.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

Two of the best studied and understood proteins are the monomeric myoglobin and the hetero-tetrameric haemoglobin (Fig. 1.5):

  • These are 3D structures solved by X-ray crystallography in the late 1950s/early 1960s2,3

  • Myoglobin has a single polypeptide chain with a haem group non-covalently associated (an organic group attached to a protein is known as a prosthetic group; Fig. 1.5a):

    • Binds O2 with a simple hyperbolic affinity curve

  • Haemoglobin has four polypeptide chains (α‎2β‎2 in adults; Fig. 1.5b), each with a haem group and very similar to myoglobin:

    • O2 affinity curve is sigmoidal due to cooperation between the four O2 binding sites. Binding of the first O2 causes an allosteric change in the protein shape that makes the binding of the next O2 easier, and so on until all four sites are occupied. See Figs 6.17, 6.18; Cellular structure and function pp.[link], [link].

Fig. 1.5 Computer-generated diagrams of (a) myoglobin and (b) haemoglobin.

Fig. 1.5 Computer-generated diagrams of (a) myoglobin and (b) haemoglobin.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

Proteins can also be modified after synthesis:

  • Post-translational modification:

    • Disulphide bonding—as described previously

    • Cross-linking—formation of covalent cross-links between individual molecules

    • Peptidolysis—enzymic removal of part of the protein after synthesis

    • Attachment of non-peptidic moieties

  • Glycosylation—addition of carbohydrate groups

  • Phosphorylation—addition of a phosphate group to specific residue(s) of a protein, e.g. serine, tyrosine

  • Adenylation—addition of an AMP group to a protein

  • Farnesylation—proteins can be attached to an unsaturated C15 hydrocarbon group (known as a farnesyl anchor) which inserts into the plasma membrane.

These modifications can have effects on the functioning of the protein, affecting, for example:

  • Regulation:

    • Phosphorylation is a common way by which protein function is modified, e.g. turning an enzyme on or off with protein kinase A (PKA). This is an example of one protein (PKA) regulating the function of others

  • Targeting:

    • Molecules conjugated to proteins can act as signals to the intracellular sorting and targeting machinery, e.g. phosphorylation can affect targeting

  • Turnover:

    • Glycosylation levels can regulate the half-life of proteins in the circulation, e.g. by affecting the rate at which they are taken up and degraded by liver cells

    • Labelling of proteins with the 74-amino acid protein ubiquitin marks them for breakdown by proteasomes

  • Structural:

    • For example, cross-linking of individual molecules in collagen greatly increases the strength of the fibril.

Structural proteins


  • Collagen is a structural fibrous protein of tendons and ligaments; it is also the protein component onto which minerals are deposited to form bone

  • There are 13 types of collagen reported to date, with type I making up 90% of the collagen found in most mammals (and up to a third of the total protein in humans)

  • Type I collagen is made up of three chains, two α‎1 and one α‎2 in a triple helix (Fig. 1.6):

    • Each individual chain is a left-handed helix formed from a repeating amino acid sequence: –Gly–Pro–X– and –Gly–X–OHPro– which occur >100 times each, accounting for ~60% of the molecule:

      • Only Gly has a small enough R group to be at the centre of such a triple helical structure

      • Collagen contains two unusual amino acids—hydroxyproline and hydroxylysine

      • Formed by post-translational enzymatic modification

    • These chains form a right-handed triple helix (tropocollagen molecule). The triple helix is stabilized by inter-chain hydrogen bonding between the peptidyl amino and carbonyl groups of the glycine and the hydroxyl groups of hydroxyproline

    • Tropocollagen molecules form bundles of parallel fibres 50nm in diameter and several millimetres long:

      • The tropocollagen molecules are staggered in their assembly

      • Tropocollagen molecules are cross-linked between lysine residues and hydroxylysine residues.

Fig. 1.6 (a) Arrangement of collagen fibrils in collagen fibres; (b) one type of cross-link formed between two adjacent lysine residues.

Fig. 1.6 (a) Arrangement of collagen fibrils in collagen fibres; (b) one type of cross-link formed between two adjacent lysine residues.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

There are clinical disorders arising from errors in collagen synthesis.

  1. 1. Osteogenesis imperfecta—there are two possible causes:

    • Abnormally short α‎1 chains: these associate with normal α‎2 chains but cannot form a stable triple helix and, therefore, nor fibres

    • Substitution of the Gly residue: replacement with either Arg or Cys also blocks formation of a stable triple helix → symptoms—brittle bones, repeated fractures, and bone deformities in children.

  2. 2. Ehlers–Danlos syndrome (Cellular structure and function OHCS11, Figs 14.6 and 14.7, p.847)—structural weakness in connective tissue.

    • Deficient cross-linking due to a defective enzyme resulting in lower numbers of hydroxylysine residues

    • Can also be due to failure to process precursor precollagen into tropocollagen → symptoms: hyperextensible skin and recurrent joint dislocation.

  3. 3. Marfan’s syndrome (Cellular structure and function OHCM10 p.706)—inherited disorder of weakened tissues.

    • Extra amino acids near C-termini of α‎2 chains results in reduced cross-linking as residues no longer line up

    • Also reduced levels of fibrillin (a small glycoprotein which is an important element of the extracellular matrix) → symptoms: weakening, especially of cardiovascular system, causing aortic rupture; skeletal and ocular muscle, lungs, and nervous systems also may be affected.


Histones are involved in the packaging of DNA (Fig. 1.7) in a space-efficient way (they increase the packing factor by ~7-fold).

Fig. 1.7 Order of chromatin packing in eukaryotes.

Fig. 1.7 Order of chromatin packing in eukaryotes.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

  • Histone proteins are globular with a cationic surface which neutralizes the phosphates of the DNA

  • Octamer of two each of H2A, H2B, H3, and H4 proteins has DNA wound around it, forming a nucleosome, which is often stabilized by the structural H1 protein:

    • Nucleosomes are further organized into helical arrays called solenoids which, in turn, are arranged around a central protein scaffold, allowing ~2m of DNA in the human nucleus to form the 46 chromosomes with a total length of just 200µm.

Concepts of biochemical reactions and enzymes

Classes of common biochemical reactions:

  • Hydrolysis: splitting with water, e.g. breaking of peptide bond

  • Ligation: joining of two compounds, e.g. two pieces of DNA

  • Condensation: forms water, e.g. synthesis of peptide bond

  • Group transfer: movement of a biochemical group from one compound to another

  • Redox: reaction of two compounds during which one is oxidized and the other reduced

  • Isomerization: physical conversion from one stereoisomer to another.

Biochemical reactions rarely occur in the absence of an enzyme.

  • An enzyme is defined as a biological catalyst:

    • Enzymes increase the rate at which a reaction occurs (usually by at least 106-fold)

    • Like a chemical catalyst, the enzyme is unchanged by the reaction that it catalyses, nor does it alter the equilibrium of the reaction (i.e. the forward and reverse reactions are speeded up by the same factor)

    • Enzymes are very specific for the reaction that they catalyse, and often enzyme activity is regulated

  • Enzymes achieve their catalytic effect by reducing the size of the activation energy step for the reaction (Gibbs’ free energy of activation or Δ‎G; Fig. 1.8):

    • Δ‎G is the difference in the free energy between the free substrates and the transition state

    • The substrates binding to the enzyme have a lower transition state energy (i.e. a lower Δ‎G) but can still react to form the same product as they could in free solution

    • → the speed of the reaction is increased.

Fig. 1.8 Enzymes decrease the activation energy.

Fig. 1.8 Enzymes decrease the activation energy.

Structure and function of enzymes

As well as being effective catalysts, one of the most striking observations concerns enzyme specificity, both in terms of the reaction catalysed and their substrates.

  • Usually, only a single reaction is catalysed (or a few very closely related reactions), with a very high, if not absolute, choice of substrate(s)

  • The active site is essential for both the catalysis and specificity of enzymes. It is defined by the structure of the enzyme protein, both at the primary level (for essential residues for the reaction) and at higher levels of protein organization (to put these residues in the correct place in the 3D structure).

Serine proteases

  • So named because they have a serine residue, which is rendered catalytically active by an aspartate and histidine residue closely adjacent in 3D space (the ‘catalytic triad’)

  • These three residues form a charge relay network on the enzyme binding a substrate (Fig. 1.9)

  • Family of enzymes includes chymotrypsin, trypsin, elastase, thrombin, and subtilisin.

Fig. 1.9 Serine proteases: schematic representations of (a) the catalytic triad; (b) the binding site and how substrate specificity is achieved.

Fig. 1.9 Serine proteases: schematic representations of (a) the catalytic triad; (b) the binding site and how substrate specificity is achieved.

Carboxypeptidase A

  • Contains a Zn molecule coordinated by two histidine residues, a glutamate, and a water molecule

  • These destabilize the substrate and allow it to be attacked by another catalytic glutamate residue, resulting in bond cleavage.


  • Cleaves glycosidic bonds between modified sugars of the polysaccharide chains that make up part of the bacterial cell wall

  • The substrate is bound in the correct orientation by a number of hydrogen bonds

  • This allows a specific aspartate residue to catalyse the cleavage reaction.

How are proteins arranged to form enzymes?

  • Enzymes can be single proteins, or multimers

  • Different tissues can have different forms of the same enzyme (isozymes); these can be used for diagnostic testing. For example, finding the heart muscle isozyme of lactate dehydrogenase (LDH; Cellular structure and function p.[link]) in the blood is indicative of a heart attack (Cellular structure and function OHCM10 pp.118, 119, 688)

  • As well as multimeric enzymes, there are also protein complexes that contain multiple enzyme activities, e.g. pyruvate dehydrogenase (PDH; Cellular structure and function pp.[link], [link]).

How are enzymes regulated?

  • Allosteric effectors bind to the enzyme at a site distinct from the active site and modify the rate at which the reaction proceeds:

    • These changes are brought about by a change in the shape of the enzyme

    • Can result either in either increased or decreased rates, e.g. PDH

  • Covalent modification, usually by addition of a phosphate group to specific residue(s)

  • Subunit dissociation, e.g. cAMP-dependent protein kinase (PKA; Fig. 1.10):

    • Consists of two catalytic (C) subunits and two regulatory (R) subunits

    • The R subunit has a pseudosubstrate site for the catalytic unit, and so in the absence of cAMP binds to and inactivates the C subunit

    • cAMP binds to the R subunit, allosterically abolishing the pseudosubstrate site so that the R subunit dissociates and leaves the C subunit free to act.

Fig. 1.10 Activation of cAMP-dependent protein kinase (PKA) by cAMP (R regulatory subunit of PKA and C catalytic subunit of PKA).

Fig. 1.10 Activation of cAMP-dependent protein kinase (PKA) by cAMP (R regulatory subunit of PKA and C catalytic subunit of PKA).

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

Enzyme co-factors

In addition to their protein subunit(s), many enzymes also have essential non-protein components known as co-factors or co-enzymes.

  • These co-factors play a vital role in the reaction catalysed by the enzyme

  • They are usually either trace elements or derivatives of vitamins.

Trace elements and enzymes

  • Small but essential amounts of minerals are absorbed from the diet and used as co-factors:

    • Zinc in lysozyme enzymes (e.g. superoxide dismutase (SOD))

    • Manganese in, e.g. isocitrate dehydrogenase in tricarboxylic acid cycle (TCA) cycle

    • Cobalt (constituent of vitamin B12) essential for methionine biosynthesis

    • Selenium in, e.g. glutathione peroxidase

    • Molybdenum in oxidation/reduction reactions

    • Copper in, e.g. cytochrome oxidase.

    • Manganese in Mn-SOD

Vitamins as precursors of co-enzymes

  • Vitamins (‘vital amines’) are small organic molecules (Fig. 1.11) that cannot be made by the body and so must be obtained from the diet:

    • In most cases, vitamins must be chemically modified to form the co-enzyme. Modifications range from minor (e.g. phosphorylation) to substantial (e.g. incorporation into much larger molecules)

    • Lack of vitamins results in metabolic disorders and diseases associated with deficiency, e.g. vitamin C (ascorbic acid) and scurvy (Cellular structure and function OHCM10 p.268):

      • Most vitamins are only required in very small amounts in the diet

      • Water-soluble vitamins are usually co-enzyme precursors.

Examples of vitamins and their active roles

  • Biotin → covalently bonded to phosphoenolpyruvate carboxykinase (PEPCK): gluconeogenesis

  • Pyridoxine (vitamin B6) → pyridoxal phosphate: transamination

  • Pantothenic acid → co-enzyme A: acyl transfer across the internal mitochondrial membrane (IMM)

  • Riboflavin (vitamin B2) → FAD: oxidation reduction, e.g. TCA cycle and electron transport chain (ETC)

  • Niacin → NAD: oxidation reduction, e.g. TCA cycle and ETC

  • Thiamine (vitamin B1) → thiamine phosphate: PDH and α‎-ketoglutarate dehydrogenase in glycolysis

  • Folic acid → tetrahydrofolate derivatives: biosynthetic reactions.

Enzyme kinetics

A number of factors can affect the rate at which enzyme-catalysed reactions proceed (Figs 1.12), including:

  • Temperature: generally, reactions proceed faster with increased temperature until a point is reached at which the crucial higher orders of protein structure are destroyed (‘denaturation’)

  • In humans, most (but not all) enzymes are most efficient at ~37°C

  • pH: the level of protonation of amino acid side-chains in proteins is dependent on the environmental pH:

    • Most cytosolic enzymes have maximal activity at pH 7.4 whereas, for example, those in the acid environment of the stomach have a much lower pH optimum

  • Amount of enzyme: the more enzyme, the more active sites and, therefore, the more reactions can be catalysed. Enzyme activity is often expressed as either the total activity (units of activity per volume of enzyme solution) or the specific activity (units per amount of protein)

  • Concentration of substrates and products: enzymes are catalysts and, as such, only increase the speed at which reactions achieve equilibrium, and do not change the equilibrium itself.

Fig. 1.12 Effect of (a) pH and (b) temperature on enzyme activity. In (a), (1) represents the majority of enzymes, and (2) gastric enzymes.

Fig. 1.12 Effect of (a) pH and (b) temperature on enzyme activity. In (a), (1) represents the majority of enzymes, and (2) gastric enzymes.

Enzyme activity can be measured by looking at the rate at which a product appears (or a substrate disappears) under defined reaction conditions:

  • The interaction between enzyme rate (V) and substrate concentration ([S]), as seen in Fig. 1.13, can be described for most enzymes by the Michaelis–Menten equation:


  • This can also be drawn as a linear plot, either a Lineweaver–Burke (1/V vs 1/[S]), Eadie–Hoftee (V vs V/[S]), or Hanes–Woolf ([S]/V vs [S]) plot

  • The Hanes–Woolf plot can be considered the most reliable as it gives greatest weight to the most robust data (i.e. at highest [S], when there is highest activity).

Fig. 1.13 Effect of substrate concentration on the reaction velocity catalysed by a classical Michaelis–Menten type of enzyme.

Fig. 1.13 Effect of substrate concentration on the reaction velocity catalysed by a classical Michaelis–Menten type of enzyme.

Useful constants that can be determined from enzyme assay studies include:

  • Km: the affinity of the enzyme binding site for its substrate

  • Vmax: the maximum rate at which the enzyme can operate (given unlimited substrate)

  • Turnover number: the number of reactions that the enzyme can perform per unit time.

Kinetic analysis allows inhibitors of enzymes to be defined into different classes:

  • Irreversible: the inhibitor covalently modifies the enzyme and so permanently inhibits it

  • Reversible: these inhibitors do not permanently affect the enzyme. These can be usefully analysed kinetically to determine whether they are competitive or non-competitive:

    • Competitive: the inhibitor and the substrate compete for the same binding site (change in kinetic parameters: Km increased, Vmax unchanged)

    • Non-competitive: the inhibitor binds at a site distinct to the substrate binding site (Km unchanged, Vmax decreased)

    • Uncompetitive: inhibitor binds at a site distinct to the substrate, but only when the enzyme also has substrate bound (Km decreases, Vmax decreases).

Fig. 1.14 (a) Plots of enzyme reaction rate in the presence of competitive and non-competitive inhibitors, shown as a double reciprocal (Lineweaver–Burke) plot and a single reciprocal (Hanes–Woolf) plot. (b) Schematic of different types of inhibition (S = substrate, I = inhibitor).
Fig. 1.14 (a) Plots of enzyme reaction rate in the presence of competitive and non-competitive inhibitors, shown as a double reciprocal (Lineweaver–Burke) plot and a single reciprocal (Hanes–Woolf) plot. (b) Schematic of different types of inhibition (S = substrate, I = inhibitor).

Fig. 1.14 (a) Plots of enzyme reaction rate in the presence of competitive and non-competitive inhibitors, shown as a double reciprocal (Lineweaver–Burke) plot and a single reciprocal (Hanes–Woolf) plot. (b) Schematic of different types of inhibition (S = substrate, I = inhibitor).

Membrane transporter proteins: structure and function

(See also Cellular structure and function pp.[link][link].) Membrane transporter proteins (Fig. 1.15) can be split into:

  1. 1. Channels: aqueous-filled pores that form a pathway for small hydrophilic substrates (usually ions) to cross lipid bilayers. The passage of substrates is nearly always gated in some way, with the channel being opened by, for example, a compound binding (ligand-gated), a change in membrane potential (voltage-gated), stretching the membrane (stretch-activated). Any of the activators result in a conformational change in the channel that allows the passage of substrate.

    • Examples: nicotinic acetylcholine receptor (nAChR, neuromuscular junction), voltage-gated Na+ channels (nerve), stretch-activated Ca2+ channels (smooth muscle).

  2. 2. Transporters.

    • Primary active—use ATP hydrolysis to energize movement of substrates:

      • Examples: Na+/K+-ATPase (aka the sodium pump), Ca2+-ATPase

    • Secondary active—use the energy from an (electro)chemical gradient set up by a primary active transporter:

      • Examples: Na+-glucose co-transporter (SGLT), Na+-Ca2+ exchange

    • Facilitated diffusion—speeds up the equilibration of substrates across cell membranes but, unlike the two previous classes, cannot concentrate substrates above equilibrium:

      • Examples: facilitated glucose transporter (GLUT).

Fig. 1.15 The main types of membrane transport systems.

Fig. 1.15 The main types of membrane transport systems.


  • Despite the wide range of functions of membrane transporter proteins, they have remarkable similarities regarding their molecular structures, although it should be noted that only a few membrane transporter proteins have been structurally characterized by X-ray crystallography and, thus, much is based on prediction

  • Both channels and transporters have multiple membrane spanning domains (transmembrane domains (TMs)), usually α‎-helixes, connected by intra- and extracellular loop regions. As the TMs pass through the hydrophobic core of the lipid bilayer, they tend to be made up of largely hydrophobic amino acids.


  • The aqueous pore in the channel is formed from a number of TMs, which have a polar amino acid residue once every turn of the α‎-helix, giving a polar side to the TM. A number of such TMs then come together, forming a pore lined with hydrophilic residues which thus allows water to enter and substrates to pass through

  • The specificity of an ion channel is often determined by the charges on residues at the ‘mouth’ of the channel. For example, the nAChR is a channel for cations and has a ring of negatively charged residues at the mouth of the channel to attract cations and repel anions:

    • Experimentally reversing these charges with site-directed mutagenesis can turn it into an anion-selective channel.


  • Many are predicted to have 12 TMs (although estimates can vary between 10 and 14)

  • Although the amino acid sequences of the many families of transporters have little in common, there are a few motifs/features:

    • Proteins with the motif for ATP binding/hydrolysis are known as ABC transporters

    • Some transporters appear to have two similar halves, suggesting they evolved from gene duplication

  • Function by binding substrate then undergoing a conformational change, resulting in the substrate-binding site being re-orientated to face the opposite side of the membrane:

    • Binding site is usually very specific for a small number of very closely related substrates

    • Binding of substrate to transporter is the basis behind transporter kinetics (Cellular structure and function p.[link]).

Examples of ABC transporters

  1. 1. ‘Flippases’: ATP-driven transporter proteins which ‘flip’ membrane lipids from one side of the lipid bilayer to the other. Roles in creating/maintaining asymmetric distribution of lipids in membranes, cellular signalling.

  2. 2. pGlycoprotein.

    • Widely expressed ATP-driven transporter with a wide substrate range of small lipophilic compounds

    • Known substrates include a large number of drug molecules, which are exported from cells and out of the body:

      • This can reduce absorption rates (intestine), increase excretion rates (liver, kidney), and affect tissue distribution (e.g. blood–brain barrier)

      • Genetic variations between individuals in pGlycoprotein expression will affect drug efficacy between patients. Gene profiling of patients may allow tailoring of drug prescribing in future (ethically contentious).

  3. 3. Cystic fibrosis (Cellular structure and function OHCM10 p.173) transmembrane conductance regulator (CFTR).

    • CFTR is the channel involved in Cl exit across the apical membrane in secretion (e.g. sweat glands, pancreatic ducts)

    • The majority of CF cases are caused by a mutation → the deletion of a single Phe amino acid (Δ‎F508). The Δ‎F508 mutant protein misfolds and fails to traffic to the apical membrane.

Roles of lipids

Lipids play a wide range of roles in the body—from energy storage to hormone signalling, plasma membrane structure to heat insulation. They are a structurally diverse class of macromolecules that have the common feature of being poorly soluble in water (hydrophobic).


  • Stored triacylglycerides (‘fat’) makes up about 20% of the body mass of an average person

  • Efficient way to store energy:

    • Higher specific energy (kJ g–1) than glycogen (Cellular structure and function p.[link]) or protein

    • Much lower hydration level than glycogen due to the inherent hydrophobicity of lipids

    • To store as much energy in glycogen would require approximately a doubling of body mass!

Structural roles

  • As discussed elsewhere, lipids (phospholipids, sphingolipids, cholesterol) are the main structural components of the lipid bilayer membranes (Cellular structure and function p.[link]):

    • The phospholipids arrange themselves into a bilayer with the polar headgroups facing outwards towards the aqueous environment and the hydrophobic ‘tails’ pointing inwards

    • The hydrophobic interior of the bilayer acts as a diffusion barrier to prevent ions or water-soluble (hydrophilic) molecules from crossing to enter or leave, e.g. the cell (plasma membrane) or organelles

    • To be able to cross the membrane, such compounds require a specific pathway (e.g. a channel or transporter, Cellular structure and function p.[link])

  • Lipids also stabilize fat–water interfaces:

    • Bile salts in the intestine: act as detergent-like compounds to emulsify dietary lipid and allow it to be absorbed as chylomicrons

    • Phospholipid in membrane: as mentioned previously, the polar headgroups allow the formation of the bilayer

    • Cholesterol in membrane: as a lipid-soluble molecule, cholesterol inserts into the hydrophobic portion of the membrane. In doing so, it makes the membrane less fluid by sterically inhibiting the movement of the fatty acyl chains

    • Pulmonary surfactant: this complex mixture of phospholipids and proteins reduces the surface tension at the interface of the alveolar lining fluid and the air in the lung, preventing alveolar collapse.

    • Premature babies (born before surfactant production begins) are unable to inflate their lungs (infant respiratory distress syndrome). Artificial surfactant can assist in these cases

    • Insulation: lipids can be either electrical insulators, as in the myelination of nerves (Cellular structure and function p.[link]), or thermal insulation, e.g. subcutaneous fat.

Signalling molecules

  • Extracellular—a number of classes of hormones are synthesized from cholesterol:

    • Steroid hormones

    • Eicosanoids (prostaglandins, thromboxanes, leukotrienes)—derived from arachidonic acid, an abundant component of plasma membrane phospholipids (e.g. as a fatty acid tail of phosphatidylcholine, phosphatidylethanolamine). Released by phospholipases A2 in response to hormone and inflammatory signals

  • Intracellular—second messengers can be derived from the breakdown of the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2). Phospholipase C cleaves the headgroup to leave two components with signalling roles:

    • Inositol-1,4,5-triphosphate (IP3) enters cytoplasm where it causes release of calcium from intracellular stores

    • Diacylglycerol (DAG) remains in the membrane:

      • Activates PKC, which has become membrane associated due to the rise in intracellular calcium

      • PKC phosphorylates target proteins, affecting their activity.

Fatty acids and triacylglycerides

Fatty acids have the general structure of a long hydrocarbon chain (usually 14–24) with a terminal carboxyl group (Fig. 1.16):

  • Most animal fatty acids have an even number of carbon atoms (Cellular structure and function p.[link])

  • Fatty acids can display different levels of saturation, depending on the number of C=C bonds in the chain.

Fig. 1.16 (a) Structure of stearic acid (C18); (b) simple representation of fatty acids.

Fig. 1.16 (a) Structure of stearic acid (C18); (b) simple representation of fatty acids.

What is generally thought of as ‘fat’ (i.e. the form that lipids are stored as in the body) are triacylglycerides (triglycerides), consisting of three fatty acids esterified to a glycerol molecule (Fig. 1.17):

  • Triacylglycerides are good for energy storage due to their high energy per gram and low hydration level

  • Major site of storage: adipocyte cells of adipose tissue.

Fig. 1.17 A triacylglycerol and its component parts.

Fig. 1.17 A triacylglycerol and its component parts.

Sources of fatty acids

Fatty acids can be either obtained from the diet or made in the body de novo:

  • Triacylglycerides from the diet are absorbed across the intestinal epithelium and transported in the blood by chylomicrons to the liver and adipose tissue, where they are hydrolysed to free fatty acids by lipoprotein lipase and cross into the cell by diffusion

  • Triacylglycerides synthesized de novo in the body (mainly in the liver) are carried by very low-density lipoprotein (VLDL) to other tissues.

Essential fatty acids

The body does not have the metabolic pathways to make all fatty acids and, therefore, some must be obtained from the diet (essential fatty acids). Mammals cannot introduce C=C bonds past C9 of the hydrocarbon chain:

  • Linoleic (C=C at C9 and C12) and linolenic acids (C=C at C9, C12, and C15) are the essential fatty acids. Good dietary source: sunflower seed oil.


Phospholipids are diacylglycerol molecules with a headgroup attached to the 3-position via a phosphodiester bond (Figs 1.18, 1.19):

  • Phospholipids are the major constituent of the plasma membrane lipid bilayer (Cellular structure and function p.[link])

  • Phospholipid is the active component of pulmonary surfactant, essential for maintenance of lung structure.

Fig. 1.18 The structure of a molecule of phosphatidic acid.

Fig. 1.18 The structure of a molecule of phosphatidic acid.

Fig. 1.19 Sample headgroup structures.

Fig. 1.19 Sample headgroup structures.

There are several different classes of phospholipids:

  • Phosphatidyl compounds—common headgroups include:

    • No headgroup except for the glycerol → phosphatidylglycerol. The phosphatidylglycerol can be further modified to cardiolipin

    • Serine → phosphatidylserine

    • Ethanolamine → phosphatidylethanolamine

    • Choline → phosphatidylcholine (lecithin)

    • Inositol → phosphatidylinositol (can be phosphorylated and plays a role in intracellular signalling—Cellular structure and function p.[link])

  • Sphingolipids have a backbone derived from sphingosine rather than glycerol:

    • Sphingosine is an amino alcohol with a long (12C) unsaturated hydrocarbon chain

    • Sphingomyelin is the only non-phosphatidyl membrane lipid; it has a choline headgroup and a fatty acid linked to the sphingosine backbone by an amide bond (Fig. 1.20)

  • Many membranes also contain glycolipids, which have a sugar unit for the headgroup:

    • In animals, glycolipids are also derived from sphingosine:

      • Cerebrosides are the simplest, having the same basic structure as sphingomyelin except with either a glucose or a galactose ester linked in place of the choline group (Fig. 1.20)

      • Gangliosides have more complex sugars as the headgroup, with a branched chain of up to seven sugar residues (Fig. 1.20).

Fig. 1.20 Diagram of a molecule of (a) sphingomyelin; (b) a cerebroside; and (c) a ganglioside.

Fig. 1.20 Diagram of a molecule of (a) sphingomyelin; (b) a cerebroside; and (c) a ganglioside.

Cholesterol and its derivatives

Cholesterol (Fig. 1.21) is a four-ring, 27C compound, synthesized from acetyl-CoA (mainly in liver). Cholesterol is an important lipid in animals, with a variety of roles:

Fig. 1.21 (a) Conventionally drawn structure of cholesterol; (b) structure drawn to indicate actual conformation of cholesterol.

Fig. 1.21 (a) Conventionally drawn structure of cholesterol; (b) structure drawn to indicate actual conformation of cholesterol.

  • Component of plasma membranes:

    • Structural role: affects the fluidity of the membrane

  • Precursor of steroid hormones:

    • All steroid hormones are synthesized by a common pathway starting with cholesterol

    • Important steroids include:

      • Sex hormones (female: progesterone, oestrogens; male: testosterone)

      • Aldosterone (regulates sodium balance)

      • Cortisol (stress)

  • Precursor of bile salts:

    • Conjugated with glycine (glycocholate) or taurine (taurocholate)

    • Secreted from liver into intestine to emulsify dietary lipid

  • Precursor of vitamin D:

    • Key role in calcium regulation

  • Component of plasma lipoproteins (chylomicrons, VLDL, intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL)):

    • Involved in transport of cholesterol between tissues

    • LDL cholesterol (Cellular structure and function OHCM10 p.690) is known as ‘bad cholesterol’ as it is associated with increased risk of atherosclerosis:

      • Leads to deposition of cholesterol in plaques which can ultimately block blood vessels. Such blockage of coronary arteries is the cause of coronary artery disease

      • Treated with HMG-CoA reductase inhibitors (‘statins’; Cellular structure and function OHPDT2 p.22).

Carbohydrates: general principles

Carbohydrates are the most abundant form of organic matter on earth:

  • Contain the elements C, H, and O

  • Produced by the fundamental pathway of photosynthesis combining CO2 and H2O:

    • All animals are ultimately reliant on this source of new organic material.

Monosaccharides (sugars) have the general formula (CH2O)n. Most common form are the hexoses (n = 6), e.g. glucose, galactose, fructose:

  • Sugars have chiral carbon atoms, so exist as stereoisomers:

    • d-glucose is the naturally occurring form, sometimes known clinically as dextrose

  • As well as stereoisomers, as sugars are cyclisized, a second asymmetrical centre is created, giving two forms:

    • The α‎ form is when the hydroxyl group is below the ring

    • The β‎ form is when it is above the ring

    • α‎ and β‎ forms can interconvert in solution (mutorotation): equilibrium 66% β‎, 33% α‎, 1% open chain.

Although sugars may be found as monosaccharides, they can also exist as:

  • Disaccharides, e.g. sucrose (glucose-α‎,1–2-fructose), lactose (galactose-β‎,1-4-fructose), maltose (glucose-α‎,1–4-glucose)

  • Small multimers (oligosaccharides), e.g. on sphingolipids, glycoproteins

  • Mainly found as large multimers (polysaccharides).

The monosaccharide building blocks are joined by glycosidic (C–O–C–) bonds (Fig. 1.22) which are formed by dehydration reactions:

Fig. 1.22 Diagrams of (a) glucose structure; (b) 1–4-glycosidic bond; (c) glycogen (or starch); and (d) cellulose.

Fig. 1.22 Diagrams of (a) glucose structure; (b) 1–4-glycosidic bond; (c) glycogen (or starch); and (d) cellulose.

  • They are named from the numbers of the C atoms in the sugars, e.g. a 1,4 bond is a joining of C1 in the first sugar to C4 in the second

  • The type of glycosidic bond can have major effects on the final structure (and thus function) of the molecule formed

  • This can be seen in the following examples of commonly occurring polysaccharides (Fig. 1.22), all made up of glucose building blocks:

    • Glycogen (animal energy storage):

      • Very large polymer of thousands of glucose monomers

      • Predominantly joined by α‎,1–4 glycosidic bonds, with branches via α‎,1–6 linkages every 8–12 residues, forming a branched tree-like structure

    • Starch (plant energy storage):

      • Mixture of two glucose polymers: amylose is a linear α‎,1–4 glucose polymer (forms a helix); amylopectin is structurally very similar to glycogen, although the branching is less frequent (every 24–30)

    • Cellulose (plant structural molecule):

      • Unlike glycogen and starch, has β‎,1–4 glycosidic bonds between the glucose monomers to form long straight chains. Chains line up in parallel, stabilized by hydrogen bonding to form fibrils and then fibres. These fibres have the high tensile strength required for their structural role in plant tissue

      • Mammals lack the enzyme to digest cellulose (cellulase) and thus it passes undigested through the intestine (known as dietary fibre). Ruminants (e.g. cows) have cellulase-producing bacteria in their digestive tracts.

Structural carbohydrates

As well as using polysaccharides for energy storage in the form of glycogen, animals also employ structural carbohydrates in the extracellular matrix. Polysaccharide units constitute 95% of proteoglycan, with the remainder being a protein backbone.

  • The polysaccharide chains are known as glycosaminoglycans (GAGs; Fig. 1.23). The chains are made up of repeating disaccharide subunits containing either glucosamine or galactosamine (amino sugars):

    • At least one of the sugars in the disaccharide has a negatively charged group (either carboxylate or sulphate).

Fig. 1.23 Structural formulae for five repeating units of important glycosaminoglycans.

Fig. 1.23 Structural formulae for five repeating units of important glycosaminoglycans.

Examples include:

  • Hyaluronic acid (hyaluronate; Fig. 1.23)

  • Chondroitin sulphate

  • Keratan sulphate (Fig. 1.24):

    • Found in cartilage extracellular matrix

    • Large (around 105kDa), highly hydrated molecule, which acts as a cushion in joints (Fig. 1.25)

  • Dermatan: found in skin

  • Heparin: present on blood vessel walls where it plays a role in preventing inappropriate blood clotting (Cellular structure and function p.[link]).

Fig. 1.24 In cartilage many molecules of aggrecan are attached non-covalently to a third GAG (hyaluronan) via link protein molecules to form a huge complex.

Fig. 1.24 In cartilage many molecules of aggrecan are attached non-covalently to a third GAG (hyaluronan) via link protein molecules to form a huge complex.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

Fig. 1.25 Large, highly hydrated complex GAG molecules form the cushion in cartilage in joints.

Fig. 1.25 Large, highly hydrated complex GAG molecules form the cushion in cartilage in joints.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

Carbohydrates as metabolic precursors

While polysaccharides play roles as energy sources and structural components, monosaccharides are also important as precursors in biosynthetic pathways.

Amino acid synthesis

  • May be made from glycolysis, pentose phosphate, or TCA cycle intermediates

  • Simplest reactions are transaminations (Cellular structure and function p.[link]) to make alanine and aspartate (from pyruvate and oxaloacetate respectively, with glutamate as the nitrogen donor and pyridoxal phosphate as the co-factor)

  • Aspartate is a precursor for other amino acids (→ asparagine, methionine, threonine (→ isoleucine, lysine))

  • α‎-ketoglutarate → glutamate (→ glutamine, proline, arginine)

  • 3-phosphoglycerate → serine (→ cysteine, glycine)

  • Pyruvate → alanine, valine, leucine

  • Phosphoenolpyruvate + erythrose-4-phosphate → phenylalanine (→ tyrosine), tyrosine, tryptophan

  • Ribose-5-phosphate → histidine.

Fatty acid synthesis

  • Fat is a major energy storage form

  • Excess carbohydrate enters the TCA cycle as normal, but as cell does not need to make ATP, the level of citrate builds up:

    • Citrate leaves the mitochondrial matrix and enters the fatty acid synthetic pathway (Cellular structure and function p.[link]).

Nucleotide synthesis

  • Nucleotides consist of a sugar moiety and a nitrogenous base

  • The sugar ribose-5-phosphate is the starting place for the synthetic pathway of the purine bases

  • In contrast, the pyrimidine base is made first, and then linked to ribose-5-phosphate

  • Deoxyribonucleotides are made by reduction

  • The ribose 5-phosphate is synthesized from glucose 6-phosphate by the pentose phosphate pathway (Cellular structure and function p.[link]).

Carbohydrates as conjugates

Many proteins and lipids have carbohydrate moieties attached, e.g. most secreted proteins (e.g. antibodies), many integral membrane proteins, and membrane lipids.


There are two ways in which carbohydrates can be attached to proteins:

  • O-glycosidic link (O-linked) to the hydroxyl group of a serine or threonine

  • N-glycosidic link (N-linked) to the amine group of asparagines:

    • Consensus sequence Asn–X–Ser or Asn–X–Thr where X is any amino acid, except proline:

      • Motif necessary but not always used due to protein 3D structure constraints

    • First two sugars added always N-acetylglucosamines, followed by three mannose residues that form a ‘core’. Addition of further monosaccharides gives rise to a huge diversity of oligosaccharide structures of two major forms:

      • High mannose—additional mannose residues added to the core described

      • Complex—variety of (less common) monosaccharide units added to core.

The wide diversity of protein-linked oligosaccharide suggests a variety of functionally important roles, although these are not well understood at present. One important role is as recognition signals, of which blood groups are a good example:

  • There are four major blood groups in humans: A, B, AB, and O (Cellular structure and function p.[link])

  • These correspond to the presence of certain oligosaccharide residues on the erythrocyte integral membrane proteins (e.g. glycophorin A):

    • There is a basic core oligosaccharide, which can have a different terminal sugar or none at all (Figs 1.26, 1.27)

  • Some individuals do not have the enzymes (glycosyl transferases) to add the terminal sugar (type O), whereas others can make type A, some type B, and some both (type AB).

Fig. 1.26 The molecular basis of ABO blood groups: (a) type A; (b) type B; and (c) type O.

Fig. 1.26 The molecular basis of ABO blood groups: (a) type A; (b) type B; and (c) type O.

Fig. 1.27 The agglutination reaction of incompatible blood types. If the blood sample is compatible, the mixed blood sample appears uniform. If the blood is incompatible with the serum, it aggregates and precipitates as shown.

Fig. 1.27 The agglutination reaction of incompatible blood types. If the blood sample is compatible, the mixed blood sample appears uniform. If the blood is incompatible with the serum, it aggregates and precipitates as shown.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p243 Oxford University Press.


The main glycolipids are based on the sphingolipids, with one or more sugars replacing the sphingosine headgroup of the phospholipids (Cellular structure and function p.[link]). There are two groups:

  • Cerebrosides: simple glucose or galactose residue. Important in brain cell membranes

  • Gangliosides: more complex branched chain of several monosaccharide groups. Also involved in the blood group types in erythrocytes.

Nucleic acids

Molecular structure of nucleic acids

DNA and RNA are nucleic acids:

  • Consist of a long polymer of nucleotides (also known as bases)

  • Nucleotides are made up from a nitrogenous base, a sugar (deoxyribose in DNA, ribose in RNA), and a phosphate group

  • There are two types of nitrogenous base (Fig. 1.28):

    • The purines: guanine (G) and adenine (A)

    • The pyrimidines: cytosine (C) and thymine (T, only in DNA) or uracil (U, only in RNA)

  • DNA has a polarity, in that it is always read 5´→3´:

    • The terms 5´ and 3´ refer to the free carbon atom from the sugar which is free at the end of the chain in question:

      • The 5´ end has a phosphate on the C5 of the (deoxy)ribose

      • The 3´ end finishes with a hydroxyl group on the C3

  • DNA strands associate into pairs and run in opposite directions (anti-parallel) (Fig. 1.29)

  • Base pairing rules are always followed:

    • C pairs with G with three hydrogen bonds

    • A pairs with T (or U in RNA) with two hydrogen bonds

  • The two strands of anti-parallel DNA form a double helix (Fig. 1.30):

    • Elucidated from X-ray diffraction images of crystallized DNA

    • Structure solved by Watson and Crick (1953).4

Fig. 1.29 Two anti-parallel strands of DNA (B = base).

Fig. 1.29 Two anti-parallel strands of DNA (B = base).

Fig. 1.30 Outline of the backbone arrangements in the DNA double helix, showing the base pairs in the centre of the helix.

Fig. 1.30 Outline of the backbone arrangements in the DNA double helix, showing the base pairs in the centre of the helix.

Reproduced with permission from Papachristodoulou, D., Biochemistry and Molecular Biology 6e, 2018, Oxford University Press.

DNA replication

DNA replication is semi-conservative (Cellular structure and function p.[link]).

Amino acid coding

The sequence of the nucleotides codes for amino acids in proteins:

  • Codons are triplets of bases that code for an amino acid

  • 43 = 64 potential triplets, so some of the 20 amino acids in proteins are coded for by more than one triplet (Table 3.1, Cellular structure and function p.[link])

  • ATG is the start codon for almost all proteins

  • TAG, TGA, and TAA are all stop codons.

The organization of cell membranes: the plasma membrane

Plasma membranes establish discrete environments (Fig. 1.31). The cell membrane bounds the cell contents; other membranes establish cell inclusions—the nucleus, mitochondria, endoplasmic (sarcoplasmic in muscle) reticulum, Golgi vesicles, lysosomes, and endosomes.

  • Membranes are fluid mosaics: there is a lipid bilayer, in which proteins are embedded

  • The fluidity derives from free movement of both lipids and proteins within the membrane

  • Membranes can be from 25% protein:75% lipid (myelinated nerves) to 75% protein:25% lipid (mitochondrial membrane)

  • Membrane proteins and lipids are often also glycosylated and contribute to the glycocalyx

  • There are three lipid classes: phospholipids, sphingolipids, and cholesterol (Cellular structure and function pp.[link][link])

  • Bipolar phospholipids are most abundant:

    • They comprise a charged headgroup and two uncharged hydrophobic tails

    • The tails face inwards, thereby forming the bilayer

    • Polar headgroups face the extra and intracellular environments

    • Lipid tails can have kinks due to double bonds

  • For most phospholipids, there are four chemical components: fatty acids, glycerol, phosphate, and one other species

  • Principal lipids are phosphatidylcholine (lecithin), phosphatidylserine, and phosphatidylethanolamine

  • Sphingolipids have a similar structure and can be glycosylated

  • Cholesterol, an unsaturated alcohol (sterol), inserts between phospholipids and influences membrane fluidity. This allows lateral diffusion of membrane proteins, important for hormone-receptor coupling.

Fig. 1.31 The structure of the plasma membrane: (a) the basic arrangement of the lipid layer; (b) a simplified model showing the arrangement of some of the membrane proteins.

Fig. 1.31 The structure of the plasma membrane: (a) the basic arrangement of the lipid layer; (b) a simplified model showing the arrangement of some of the membrane proteins.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p20 Oxford University Press.

Membrane composition is asymmetrical: the inner half of the bilayer contains more phospholipids with negatively charged headgroups (e.g. phosphatidylserine). A ‘flippase’ enzyme (an ABC transporter family member, Cellular structure and function p.[link]) uses ATP as an energy source, sweeping the membrane to preserve asymmetry. Asymmetry maintains cell shape (although proteins and the cytoskeleton also play a major role) and ensures correct association of particular proteins with certain lipid species.

Membrane proteins can be integral or extrinsic (or peripheral).

Integral proteins

  • Have a variety of functions:

    • Receptors—transmembrane proteins which bind ligands, undergo conformational changes to:

      • Initiate enzyme activity on an intracellular domain

      • Open a pore through the protein

      • Activate intracellular signalling cascades

    • Transporters: channels or carriers

    • Enzymes

    • Adhesion molecules: integrins for extracellular matrix, cadherins for cells

  • Can span the membrane once or may have multiple, closely packed transmembrane segments (e.g. ten spans in the Na+-K+-ATPase). Transmembrane spans are typically 25 amino acid α‎-helices—hydrophobic residues face the lipid environment of the membrane

  • In some cases can only partially cross the membrane or sit at the extra or intracellular faces and link to phospholipids by oligosaccharides (GPI-linked proteins)

  • Can be multimers made of non-covalent bound subunits. Multimers may comprise multiple copies of a single protein or two or more different ones

  • Can combine in non-mobile clusters or act as anchors for non-covalent binding of extrinsic cytoskeletal proteins.

Extrinsic proteins

  • Form non-covalent bonds with integral proteins

  • May be components of the cytoskeleton (e.g. spectrin, ankyrin), that bind transmembrane channels, carriers, and adhesion molecules. They define cell shape, strength, and polarity.

Transport across membranes

Membranes form selective barriers to maintain the composition of different compartments. Without proteins, only small non-polar solutes would permeate the lipid bilayer. Even when lipid permeability exists, movement can be augmented by protein pathways:

  • The simplest pathway is solubility diffusion through lipid

  • Gases such as oxygen can dissolve in the lipid and then diffuse passively through Brownian motion along concentration gradients according to Fick’s law: J= –D × A × ∆C/∆x

    where     J = diffusion flux

                D = diffusion coefficient of solute

                A = surface area of membrane

                Δ‎C = concentration gradient

                Δ‎x = membrane thickness

    • Diffusion is a passive process: there is no direct energy expenditure involved

  • Water also diffuses across membranes by osmosis, from regions of high water concentration (hypo-osmotic) to regions of low water concentration (hyperosmotic):

    • Although water can dissolve in and diffuse through lipids, most cell membranes contain selective aquaporin proteins that convey a high permeability to water and mean that osmotic gradients cannot be sustained. Water movements cease when intracellular and extracellular solutions become isotonic.

Other solutes, including ions and some organic osmolytes such as taurine also move across the membrane by passive diffusion through channels which are water-filled protein pores:

  • Channels can be constitutively open (leak channels) or gated, i.e. opened by:

    • Change in membrane potential

    • The extracellular binding of a ligand to the channel itself or to a G-protein-linked receptor associated with the channel

    • The intracellular binding of a second messenger (such as cGMP)

    • Membrane deformation

  • Channels can be selective for the ions that can permeate. There are families of channels specific for ions including Na+, K+, Ca2+, and Cl

  • Gap junctions are channels connecting cytoplasm of two cells. Each membrane contains connexons, comprising six connexins which form a pore. The two pores are aligned to make a patent, non-selective channel for electrical and chemical cell–cell communication.

Other transport processes are mediated by carrier proteins (Fig. 1.32):

  • Carriers undergo a conformation change to present the solute at the opposing face. The binding and conformation change processes mean that the process is:

    • Slower than channel-mediated transport

    • Temperature sensitive

    • Saturable, with Vmax and Km

  • Passive carriers mediate facilitated diffusion, e.g. the GLUT carrier for glucose:

    • Like simple diffusion, there is no direct input of energy and the solute gradient is dissipated by the transport process

    • Movement of solute stops when equilibrium is reached. For uncharged solutes, this is when the inward and outward chemical gradients are equal. Charged solutes reach equilibrium when the electrochemical gradients are equal, although an asymmetry of solute concentration at either side of the membrane will still exist.

Fig. 1.32 The main types of carrier proteins employed by mammalian cells.

Fig. 1.32 The main types of carrier proteins employed by mammalian cells.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p36 Oxford University Press.

Active transport processes also exist. These accumulate the transported solute to a level above that predicted by passive equilibration:

  • Active transporters undergo conformational changes energized directly or indirectly by ATP hydrolysis

  • Primary active transporters are ATPases—the hydrolysis of ATP by the protein initiates a conformational change that translocates bound ions across the membrane:

    • For example, Na+/K+-ATPase, keeping intracellular [Na+] low in cells, the Ca2+-ATPase, which extrudes Ca2+ and the H+/K+-ATPase in the stomach, which secretes gastric acid

  • Secondary active transporters use gradients made by primary systems to energize transport of other solutes. An inward Na+ electrochemical gradient is most commonly used. A conformational change upon solute binding translocates the two solutes across the membrane. The driver ion gradient dissipates as it energizes accumulation of the substrate. The process can be:

    • Co-transport (symport), e.g. Na+-glucose co-transport by the SGLT protein

    • Exchange (antiport), e.g. Na+ × H+ exchange by the NHE protein.

Intracellular organelle membranes also possess transport pathways. Notable examples include H+-ATPases in lysosomes, Ca2+-ATPases in mitochondria and in endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR), and Ca2+ channels in ER/SR.

The intracellular milieu

In an average 70kg adult, total body water volume is 42L: there are 14L of extracellular fluid and 28L of intracellular fluid. The intracellular and extracellular compartments differ markedly in their composition, and it is the cell membrane separating the compartments that is responsible for the maintenance of the differences.

Extracellular fluid comprises:

  • Blood plasma - 3L

  • Interstitial fluid (including bone and connective tissues) - 10L

  • Transcellular fluid (considered to be fluid found in enclosed epithelial-lined cavities; for example, synovial fluid, cerebrospinal fluid, pleural fluid, ocular fluid) - 1L

Plasma and interstitial fluid are essentially identical with the exception of proteins, which are unable to exchange across capillary walls and so are largely absent from interstitial fluid. The composition of transcellular fluid is variable.

Typical values for the composition of the extracellular and intracellular compartments are listed in Table 1.2.

Table 1.2 Typical extracellular and intracellular compartment composition values




















1–2mM (10–7M free)



18mM (1mM free)




  • The most important difference is the separation of Na+ and K+ ions:

    • The Na+-K+-ATPase membrane transporter protein actively extrudes Na+ ions from and accumulates K+ ions in the cell.

    • The asymmetry underlies the establishment of the resting membrane potential and the generation of action potentials in excitable cells

  • Free [Ca2+] is kept low inside cells by active extrusion of Ca2+ ions by a Ca2+-ATPase and by secondary active Na+ × Ca2+ exchange, and sequestration within the ER by another Ca2+-ATPase (SERCA):

    • Low steady-state [Ca2+] levels can be exploited for cell signalling, when hormones initiate second messenger cascades, which liberate Ca2+ from intracellular stores

  • Intracellular pH is maintained close to neutrality by active extrusion of H+ ions by a primary H+-ATPase or, more commonly, by secondary active Na+-H+ exchange. Na+-driven HCO3 uptake can also contribute to acid extrusion by adding HCO3 ions to buffer the cytoplasm:

    • Without these active acid extruders, the negative membrane potential would mean that H+ ions would be passively equilibrated at approximately pH 6.6

  • Cl ions are accumulated inside cells by Na+-driven active processes, although the effect is restricted by passive efflux of Cl through channels

  • A large proportion of intracellular osmotic potential is derived from impermeant structural proteins, the concentration of which can be 300g L–1:

    • These ensure that the intracellular solution, like the extracellular solution, has similar numbers of anions and cations, and hence exhibits bulk electroneutrality (despite varying contributions from different ions)

  • The high water permeability of the cell membrane that results from water channels (aquaporins) means that cell volume must change in response to changes in extra- or intracellular osmolarity:

    • Extracellular osmolarity is tightly regulated, although there are regions in the body—notably in the kidneys—where cells can be subjected to varying osmotic conditions

    • Intracellular osmolyte content can vary with metabolic changes or following uptake or loss of solutes across the cell membrane

    • Cells can limit these changes by activating channels and carriers to lose or gain solutes and hence water. These systems rely on ion gradients established by the Na+-K+-ATPase.

Cell signalling pathways

Cell function can be regulated by a variety of external chemical factors that interface with the cell at receptors on the cell membrane or, if lipid soluble, at cytoplasmic receptors.

  • These factors include:

    • Peptides such as antidiuretic hormone (ADH)

    • Amines such as adrenaline (epinephrine)

    • Steroids such as aldosterone

    • A diverse array of small signals (nucleotides, ions, gases)

  • These regulators can be derived from:

    • The cell itself (termed autocrine regulation)

    • Nearby cells (paracrine regulation)

    • Cells located at some distance and delivered by the bloodstream (endocrine)

    • From nerve endings (neurocrine).

The agonist or ligand binds to the receptor protein and initiates a conformational change in the protein. Thereafter, a number of direct or indirect responses can be elicited (Fig. 1.33):

Fig. 1.33 The principal ways in which chemical signals affect their target cells. Examples of each type of coupling are shown. (R = receptor; E = enzyme; G = G-protein; + indicates increased activity; – indicates decreased activity.)Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p53 Oxford University Press.

Fig. 1.33 The principal ways in which chemical signals affect their target cells. Examples of each type of coupling are shown. (R = receptor; E = enzyme; G = G-protein; + indicates increased activity; – indicates decreased activity.)Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p53 Oxford University Press.

  • Ligand-gated ion channels (ionotropic receptors) are receptor proteins that operate as ion channels upon occupancy. Exemplified by the nicotinic receptor of skeletal muscle cells, which operates as a cation channel when acetylcholine binds

  • Catalytic receptors act as enzymes or are associated with enzyme complexes which are activated upon occupancy:

    • Insulin and many growth factors initiate serine/threonine or tyrosine kinases within the receptor protein, or associated with it, in this way

    • Alternatively, receptor guanylate cyclases (e.g. the ANP receptor) can convert GTP to cGMP which, in turn, activates PKG (a serine/threonine kinase)

  • Receptors can also be coupled, through a GTP-binding protein (G-protein), to effectors:

    • A G-protein is a heterotrimeric complex (Fig. 1.34). Agonist binding promotes interaction of the receptor with a G-protein, which undergoes a conformational change. GTP binding ensues, which causes the trimer to split into α‎ and β‎γ‎ units

    • G-protein-linked receptor occupancy can initiate stimulatory (GS) or inhibitory (GI) effects, largely through α‎ subunit actions on effector enzymes to generate second messenger signalling molecules:

      • Adenylate cyclase: converts ATP to cAMP. cAMP activates PKA, which phosphorylates serine and threonine residues on proteins to alter their conformation and modulate their function. Effects are reversed by phosphatases that dephosphorylate target proteins. The activation of glycogenolysis by adrenaline is mediated by PKA activation of phosphorylase kinase

      • Phospholipase C: converts membrane phosphatidyl inositols to IP3 and diacylglycerol (DAG). IP3 is released into the cytoplasm where it binds to a receptor on the ER, which acts as a channel for release of Ca2+ ions into the cytoplasm. Ca2+ exerts its effects through Ca2+-binding proteins such as calmodulin which activate serine/threonine kinases. Myosin light chain kinase can be activated in this way. DAG remains in the membrane where it associates with cytoplasmic PKC, a Ca2+-dependent serine/threonine kinase, and increases its affinity for Ca2+. PKC mediates growth factor actions such as cell shape changes and proliferation

      • Phospholipase A2 (PLA2): converts membrane phospholipids to arachidonic acid. Agonists activating PLA2 include serotonin and glutamate. Arachidonic acid is a precursor for eicosanoids. The eicosanoids are involved in inflammatory responses and modify blood vessel diameter, platelet activity, and cell membrane permeability. Cyclo-oxygenase (COX; inhibited by aspirin) generates prostaglandins, prostacyclins, and thromboxanes; 5-lipoxygenase generates leukotrienes

    • After interaction with effectors, the G-protein trimer is reformed by α‎-catalysed hydrolysis of GTP to GDP, which is displaced for GTP when an agonist–receptor complex next interacts

    • Each subunit exhibits a number of isoforms, with differences in tissue distribution and function apparent. The huge variety of subunit combinations which can be assembled provides for enormous diversity in signalling possibilities

    • G-proteins can also have direct actions on effectors: an α‎ subunit directly mediates the activation of l-type Ca2+ channels following β‎-adrenoreceptor occupancy in the heart. Likewise, a β‎γ‎ subunit complex can directly activate the muscarinic M2 acetylcholine receptor-linked K+ channel in the heart

    • A family of small G proteins also exists, which are structurally similar to the α‎ subunit. The Ras isoforms regulate cell growth by regulating kinase cascades from the cell membrane to the nucleus. Mutations in Ras lead to oncogenes which encode constitutively active Ras pathways and induce malignant transformation of the cell

  • Intracellular or nuclear receptors. These proteins bind to lipid-soluble agonists (e.g. steroid hormones such as aldosterone) which can cross the plasma membrane. Receptors may be located in the cytoplasm or in the nucleus. Cytoplasmic ones translocate to the nucleus after receptor occupancy and dissociation from heat shock chaperone proteins. The receptor conformational change ultimately leads to a change in DNA conformation which initiates transcription; responses are therefore slower than those of other receptor types.

Fig. 1.34 Receptor activation of heterotrimeric G-proteins leads to activation of enzymes and ion channels.

Fig. 1.34 Receptor activation of heterotrimeric G-proteins leads to activation of enzymes and ion channels.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p54 Oxford University Press.

Epithelial structure

Epithelia are planar sheets of cells and associated connective tissue, the precise arrangement and microstructure of which varies according to location and specialization. Epithelia:

  • Form a complete covering for the internal and external surfaces of the body (including invaginations of the body surfaces such as sweat glands and the exocrine pancreas)

  • Separate the external environment from the internal milieu

  • Form the major functional component of several organs (e.g. gastrointestinal (GI) tract, urogenital system, skin)

  • Their principal function is to mediate the selective transfer of substances and, in so doing, control the composition of the internal environment

  • Possess cells that have discrete apical (external) and basolateral (internal) membranes, which give the tissue polarity

  • May be classified according to function:

    • Some mediate secretion (e.g. sweat glands), some perform absorption (e.g. the epithelium of the GI tract), others fulfil a barrier role (e.g. skin)

    • Most epithelial cells exhibit more than one of the earlier listed three functional characteristics and certain specialized epithelia also play a role in sensation and movement (mucociliary escalator).

Epithelial cells are invariably associated with a connective tissue layer at their basal surface which they synthesize. This basement membrane contains specialized collagen protein (type IV) and provides structural integrity to the epithelial cell layer above. In certain locations, the basement membrane itself is highly specialized (e.g. in the glomerulus of the kidney).

Historically, epithelial cells were classified according to their sectional appearance under a light microscope:

  • Squamous: cells are flat

  • Cuboidal: cells are approximately square in section

  • Columnar: cells significantly taller than they are wide.

Epithelia are arranged into single or multiple layers of cells:

  • Simple epithelium: single layer of epithelial cells

  • Stratified: several layers of cells

  • Pseudo-stratified: single layer of cells which has the appearance of a stratified epithelium since nuclei are arranged at different heights.

Such distinctions are still in use, but they only provide limited information as to the functional specializations of different epithelia.

The arrangement of epithelial cells into layers accounts for many of their functional specializations. This arrangement is maintained by cell junctions between epithelial cells which bind them together and provide epithelia with their gross structure. Broadly speaking, three types of cell junction between epithelial cells exist:

  • Occluding or tight junctions: contribute to the barrier function of epithelia by linking individual cells tightly together. They also maintain the polarity of epithelial cells by separating the apical and basolateral membranes. They are formed from membrane proteins (occludins and claudins) that adhere epithelial cells together near to their apical surfaces. The extent to which they do this varies between epithelia and determines whether an epithelia is ‘tight’ or ‘leaky’

  • Anchoring junctions: provide epithelia with the ability to resist shearing and tensile forces by linking cytoskeletal elements between epithelial cells as well as to the extracellular matrix. In this way they bind epithelial cells and associated extracellular matrix together into a functional unit. Anchoring junctions comprise a component of the cytoskeleton, a cytoplasmic link-protein, and a cell–cell adhesion molecule bound in series:

    • Adherens junctions: bind the actin networks of epithelial cells together via catenins (link-protein) and cadherins (cell–cell adhesion molecule). They are particularly associated with columnar epithelial cells, where they form an adhesion belt

    • Desmosomes: connect cytoskeletal intermediate filaments between adjacent epithelial cells. The cytoplasmic link proteins are desmoplakins and the cell–cell adhesion molecules are desmogleins. The formation of antibodies to desmosomal proteins results in widespread blistering of the skin and mucous membranes—an autoimmune condition known as pemphigus

    • Hemidesmosomes: connect intermediate filament networks to the extracellular matrix. They resemble desmosomes except that instead of desmogleins, desmoplakins are bound to connective tissue receptors called integrins. They are important for the attachment of epithelial cells to the underlying basement membrane

  • Communicating (gap) junctions: mediate cell–cell communication by allowing selective diffusion of small molecules and ions between adjacent cells. They comprise proteins called connexins, which combine to form a conducting pore (connexon) in cell membranes. Connexons in adjacent epithelial cells align, allowing direct cell–cell communication. In epithelia, they are particularly important in embryogenesis, where they play a role in organization of developing sheets of cells. Gap junctions are regulated by intracellular pH and Ca2+.

Often, several types of epithelial junction are found in close proximity to each other. This is known as a junctional complex.

Epithelial cell specializations

Structural cell surface specializations occur on certain epithelial cells, normally to increase the surface area of the cell or to move foreign particles:

  • Cilia: long cytoplasmic extensions (5–10µm long, 0.25µm diameter) from the surfaces of some epithelial cells. They are motile and are important in moving fluid over cells. They are particularly important in respiratory epithelia

  • Microvilli: small, cytoplasmic projections (1µm long, 0.08µm wide), found on the cell surface of certain epithelial cells. In the intestine, for example, the mass of microvilli on the cell surface forms a brush border, aiding the absorption of nutrients

  • Basolateral folds: deep folding of the basal or lateral surface of the epithelial cells. Important in fluid or ion transport functions of cells (e.g. renal tubular cells).

Epithelial function

Epithelia are layers of cells that isolate the internal from the external environment. They regulate the movement of solutes and water to and from the body. Examples include the skin, the linings of the respiratory tract, alimentary canal, and kidney tubules.

Epithelia can be divided into two categories:

  • Absorptive: active Na+ transport drives solute and water reabsorption

  • Secretory: active Cl transport drives fluid secretion.

The cells of epithelia exhibit considerable diversity of form and function but have fundamental properties in common. They are formed into sheets, which may be multilayered, held together by tight junctions at their luminal edge. They are separated from neighbouring cells by lateral intercellular spaces.

Vectorial transport

  • The ability to translocate ions from one compartment to another (unidirectional transport) is the cardinal property of epithelia

  • It is achieved by asymmetry of the cell membranes at the two faces. The cells are termed ‘polarized’ (but not in the sense that a nerve cell is ‘polarized’):

    • The external-facing membrane is the apical (or luminal or mucosal) membrane

    • The internal-facing membrane is the basolateral (or contraluminal or serosal) membrane

  • Membranes have different morphology (villi), biochemistry (protein distribution), and function (ion selectivity), and remain separated by the tight junctions which form a barrier (to varying degrees) to solutes and water.

Tight and leaky epithelia

Epithelia can be tight or leaky (Table 1.3):

  • In tight epithelia, tight junctions prevent significant movement of molecules between cells

  • In leaky epithelia, the tight junctions form imperfect seals and are a low-resistance, leak pathway (‘shunt’) for ions and water:

    • Leaky epithelia perform bulk handling of isosmotic solutions (either for absorption or secretion) = ‘valves’. Located in proximal parts of the kidney and GI tract (e.g. proximal tubule, small intestine)

    • Tight epithelia withstand large osmotic gradients; they are more selective in the way they handle the load with which they are presented. Located distally (e.g. collecting duct, colon)

  • By placing the two different types of epithelia in series, bulk absorption, followed by fine control, is achieved.

Table 1.3 Tight vs leaky epithelia



Tight junctions



Paracellular ion permeability



Electrical resistance



Transepithelial p.d.

High (30mV)

Low (5mV)

Water permeability



Apical entry of sodium



* May be raised in collecting duct by ADH-induced insertion of water channels.

Epithelial solute transport can be transcellular (though the cells) or paracellular (between the cells). The former is ultimately dependent on active transport processes, while the latter occurs passively, by diffusion or convection.

Direction of transport depends on electrical and chemical gradients for ions, and osmotic and hydrostatic pressure gradients for water. Ion movements lead to charge separation—establish a potential difference (p.d.) across the epithelium. Orientation of p.d. depends on which ions move, and in which direction. Magnitude of p.d. depends on whether the epithelium is leaky (so that charge dissipates or ‘shunts’).

Tight epithelia have low water permeability: it may be upregulated by channel insertion (e.g. collecting duct). The water permeability of leaky epithelia is high and unregulated, and can be transcellular—through water channels (aquaporins)—or paracellular. Note that high water permeability does not necessarily derive from the leaky tight junction.

Absorptive epithelia

  • In absorptive epithelia, active transport of Na+ ions is the fundamental event

  • Described by the Ussing model (Fig. 1.35):

    • Na+ concentration is kept low in cells by the basolateral Na+-K+-ATPase

    • Na+ ions move down an electrochemical gradient into the cell across the apical membrane

    • The transepithelial movement of Na+ ions leaves the lumen negative with respect to the contraluminal side

  • The vectorial transport of sodium and associated solutes depends on the specific permeability properties of individual membranes, which are in turn determined by a ‘pick and mix’ from a selection of transport proteins

  • Basolateral membranes have properties in common with ‘regular’ cells (nerve, muscle) and possess:

    • Na+-K+-ATPase

    • K+ leak channels = high permeability to K+ (PK)

    • Low permeability to Na+ (PNa)

    • Ca2+-ATPase

    • Cl-HCO3 exchanger (‘band 3’)

    • Hormone receptors

  • Apical membranes all have high PNa:

    • In tight epithelia, this is due to Na+ channels (ENaC), inhibited by amiloride and regulated by aldosterone

    • In leaky epithelia, carrier-mediated symports and antiports are also present, appropriate for specific tissue functions (e.g. Na+-glucose for sugar absorption in small intestine)

    • Passive movement via paracellular route down electrochemical gradients established by sodium-coupled movements also occurs in leaky epithelia

  • Note that all absorptive processes depend in some way on the sodium gradient, established by the Na+-K+-ATPase in the basolateral membrane.

Secretory epithelia

In secretory epithelia (e.g. lung, pancreas), the underlying process is active Cl transport:

  • Cl ions enter the epithelial cells across the basolateral membrane on a carrier: often Na+-K+-2Cl symport

  • Cl ions accumulate within the cell and exit passively down a chemical gradient through Cl channels (which are incorrectly inserted or regulated in cystic fibrosis)

  • The exit of Cl sets up a Cl diffusion potential across the membrane = lumen negative

  • Na+ ions move passively across the epithelium via the paracellular pathway, driven by the transepithelial p.d.

  • H2O follows along an osmotic gradient

  • Note that this process is, like absorption, is dependent on the asymmetrically distributed Na+-K+-ATPase.

Organelles: overview

Eukaryote cells are distinguished from prokaryotic bacterial cells by the presence of organelles which are distinct membrane-bound compartments within the cell:

  • 90% of total cell membrane is intracellular

  • Many important metabolic reactions occur within or on these organelle membranes.

Metabolic compartmentalization is an essential role of the organelles, allowing both oxidative and reductive conditions in the cell, separating anabolic and catabolic reactions, and containing toxic compounds to prevent widespread cellular damage.

Major organelles in the cell include:

  • Nucleus:

    • Contains the genetic material (chromosomes) encased in a double membrane perforated with nuclear pores which allow movement of macromolecules in and out of the nucleus

    • Also contains the nucleoli, assemblies of RNA and protein involved in ribosome production

  • Mitochondria:

    • Responsible for the production of ATP—the cellular fuel

    • Bounded by a double membrane. The inner membrane is highly folded into cristae (increases surface area)

  • Endoplasmic reticulum (ER)—interconnected tubular membranes (cisternae) in the cytoplasm:

    • Rough ER (RER):

      • Generally, found closer to the nucleus

      • The ‘rough’ refers to the ribosomes, which give a studded appearance and are actively involved in protein synthesis

    • Smooth ER:

      • Involved in packaging and delivery of proteins to the Golgi apparatus

      • Site of membrane lipid synthesis

      • Contains cytochrome P450, which plays a role in detoxification of drugs and toxic compounds, especially in liver (Cellular structure and function p.[link])

  • Golgi apparatus:

    • Along with RER, a prominent Golgi is associated with actively secreting cells

    • Consists of a stack of flattened membrane bound vesicles, which can be distinguished into cis-, median-, and trans-Golgi regions

    • As proteins made in the RER pass through the Golgi complex, they are modified and processed (e.g. glycosylation) before entering the trans-Golgi network (TGN) for sorting and delivery to the appropriate target

  • Ribosomes:

    • Protein and RNA aggregates that catalyse the manufacture of proteins

    • Many are found attached to the RER for making secreted proteins; plus there are also free cytoplasmic ribosomes

  • Lysosomes:

    • Bounded by a single membrane and containing lytic enzymes

    • Involved in digestion of ingested macromolecules and turnover of intracellular components

  • Peroxisomes:

    • Contain enzymes involved in oxidative metabolism which use molecular oxygen and generate toxic hydrogen peroxide (H2O2)

  • Cytoskeletal elements—different types of contractile proteins:

    • Microfilaments:

      • Mostly made up of actin with regulatory proteins

      • Involved in cell movement

    • Intermediate filaments:

      • Form α‎-helical structures

      • Contribute to mechanical strength and stability of cells

    • Microtubules:

      • α‎- and β‎-tubulins forming hollow tubes

      • Involved in chromosome separation in cell division (mitosis), intracellular transport of vesicles and organelles, and movement of cilia.

NB: organelles are not independent of each other. Indeed, there is a large amount of vesicular movement between organelles, especially between the ER, the Golgi, and the TGN involved in protein trafficking.

The nucleus

The presence of a nucleus defines a cell as eukaryotic, and thus all cells in the human body have a nucleus, except mature red blood cells, which are enucleate (although the precursor cells they are derived from are nucleated).

Features of the nucleus

  • Approximately 7–8µm in diameter

  • The nuclear contents are kept separate from the cytoplasm by the nuclear membrane. This double membrane contains pores that allow macromolecules to cross. This is a two-way process. For example, nucleotides need to enter the nucleus, and messenger RNA (mRNA) leaves the nucleus to be translated in the cytoplasm:

    • The space between the inner and outer membrane is called the perinuclear space

    • The inner nuclear membrane (the nuclear lamina) consists mainly of a scaffold-like network of protein filaments (lamins or intermediate filament type V):

      • Proposed roles of the nuclear lamina include maintenance of nuclear shape and spatial organization of nuclear pores, as well as in transcription and DNA replication

  • The nucleus contains 46 chromosomes:

    • DNA–protein complexes representing the hereditary material

    • 22 homologous pairs, plus the sex chromosomes (XX = female, XY = male)

    • The chromosomes are only visible as distinct entities during cell division (Cellular structure and function mitosis p. 78 (cell cycle) or meiosis p. 86 (meiosis)), when maximally condensed. The rest of the time they are unwound and dispersed (chromatin):

      • Heterochromatin (appears darker)—relatively more condensed form

      • Euchromatin (lighter region) is less dense and contains most of the active genes

      • The Barr body is the inactive X-chromosome in female cells which appears as a darkly stained mass of chromatin

  • There are also several nucleoli, which are dense regions of RNA and protein (nucleoprotein):

    • These are regions involved in the production of new ribosomes for export into the cytoplasm through the nuclear pores

    • They are associated with particular chromosomes which have the genes for ribosomal RNA (in humans—chromosomes 13, 14, 15, 21, and 22).

Functions of the nucleus

  • Gene replication and repair:

    • As well as DNA replication during cell division, there are also mechanisms to repair DNA to maintain the integrity of the hereditary material

  • Genetic transcription:

    • Production of mRNA that will be translated into proteins in the cytoplasm by ribosomes

  • Ribosome production:

    • Production of ribosomes in the nucleoli.

Gene replication

DNA replication needs to occur before cell division (Cellular structure and function mitosis p. 78) so that each daughter cell has a complete set of hereditary material. It is obviously important that the process should be highly faithful.

  • Newly synthesized DNA requires packaging into nucleosomes and chromosomes

  • Thus DNA replication also requires significant protein synthesis

  • DNA synthesis requires a supply of nucleotides bases—the dNTPs:

    • Anti-cancer drugs (Cellular structure and function OHCM10 p.376) (e.g. methotrexate, 5-fluorouracil) stop cell division by interfering with dNTP supply.

DNA replication occurs by a semi-conservative process

  • Each daughter molecule has one DNA strand from the parent and one newly copied strand

  • Shown by Meselson and Stahl5 in a classic experiment (Fig. 1.36):

    • Escherichia coli were grown in media containing 15N, which was incorporated into their DNA

    • These cells were suddenly switched into 14N-containing media. First-generation cells had DNA which was 50% 15N, 50% 14N.

Fig. 1.36 Demonstration of semi-conservative DNA replication by Meselson and Stahl.

Fig. 1.36 Demonstration of semi-conservative DNA replication by Meselson and Stahl.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p20 Oxford University Press.

Mechanism of DNA replication

  • Helical double-stranded DNA has to be unwound by DNA helicase:

    • DNA gyrase (a topoisomerase enzyme) stops unwound single DNA strands from getting tangled by a breaking and rejoining mechanism

    • Single-stranded DNA binding proteins bind to and stabilize the single strands

    • There are now two strands: the leading and the lagging strand

  • DNA is copied by DNA polymerases in a 5′→3′ direction (Fig. 1.37):

    • These enzymes cannot initiate a new strand of DNA

    • RNA primase (an RNA polymerase) makes a short RNA primer (10–20 bases)

    • For the leading strand, DNA polymerase can extend this directly 5′→3′

    • However, the lagging strand has a 3′→5′ direction and cannot be copied directly:

      • It is copied in small pieces (Okazaki fragments), each primed with RNA

      • When the DNA polymerase encounters the RNA of a previously made fragment, a 5′→3′ RNase H removes the RNA and DNA polymerase replaces it with DNA

      • DNA ligase joins the two adjacent DNA fragments together

  • DNA polymerases use one strand of the original DNA as a template, sequentially adding the correct nucleotide using the base pair rules (C&G, A&T). These pairings need to very accurate for faithful DNA replication:

    • DNA polymerases have a proof-reading facility to enhance accuracy.

Fig. 1.37 Diagram of a replicative fork. The leading strand is synthesized continuously, while the lagging strand is synthesized as a series of short (Okazaki) fragments.

Fig. 1.37 Diagram of a replicative fork. The leading strand is synthesized continuously, while the lagging strand is synthesized as a series of short (Okazaki) fragments.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p20 Oxford University Press.

DNA damage and repair

  • Factors such as UV light and chemical agents can damage DNA, → modified bases and/or base mismatches

  • Enzymes recognize damage and repair it by nicking the strand (endonuclease), removing the incorrect/damaged bases (exonuclease), filling in the gap (DNA polymerases), and then joining up the strand (DNA ligase).


All protein synthesis takes place on ribosomes in the cytoplasm (either free or associated with RER), yet the final destination of proteins is very varied (e.g. secreted, integral membrane, intracellular organelles, cytoplasmic). Trafficking is the term given to the movement of compounds from their site of manufacture to their target site (Fig. 1.38).

Fig. 1.38 Overview of protein trafficking: how proteins are secreted from cells and how enzymes are delivered to lysosomes.

Fig. 1.38 Overview of protein trafficking: how proteins are secreted from cells and how enzymes are delivered to lysosomes.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p20 Oxford University Press.

Vesicle trafficking routes

  • Proteins which are destined for secretion, insertion into the membrane, or targeting to organelles (e.g. lysosomes) are made on the RER:

    • Those which are destined for secretion or an intraorganelle space have a signal sequence at their N-terminus which allows them to enter the RER lumen

    • Integral membrane proteins also have a signal sequence and are inserted into the RER membrane

    • Often, the signal peptide is cleaved after translation

  • Vesicles bud off from the RER and move into the Golgi apparatus, where processing and glycosylation takes place

  • After passing through the three parts of the Golgi (cis-, median-, and trans-regions), vesicles again bud off and enter the TGN, where they are sorted and targeted to the correct destination (e.g. plasma membrane, lysosomes)

  • The arrival and fusion of the vesicle at the plasma membrane either adds the new proteins to the membrane or allows the contents of the vesicle to be released into the extracellular medium. There needs to be membrane retrieval at the same rate as addition, otherwise the cell would increase in size

  • Proteins also can enter the nucleus and mitochondria, but do so after synthesis:

    • These are synthesized in the cytoplasm on free ribosomes

    • Signal sequences allow proteins to enter the mitochondria post-translationally

    • Nuclear proteins can enter via the nuclear pores—again a signal sequence is responsible for the targeting

  • Chaperonins are required for assembly of large oligomeric proteins into functional active complexes in mitochondria and ER lumen

  • Examples of inherited disorders of trafficking:

    • Lysosomal storage diseases → secretion of harmful degradative enzymes into the bloodstream rather than targeting into lysosomes (e.g. I-cell disease)

    • Primary hyperoxaluria (Cellular structure and function OHCM10 p.118) → peroxisomal enzyme mistargeted to mitochondria, resulting in an inability to metabolize oxalate.


There are two basic types:

  • Constitutive—where the proteins are secreted as soon as they are synthesized and processed

  • Regulated—proteins are synthesized, processed, and stored in vesicles before being released when a particular signal is received.

Receptor-mediated endocytosis

  • Macromolecules which cannot pass through the plasma membrane may enter cells through receptor-mediated endocytosis (Fig. 1.39):

    • Receptors are present in the plasma membrane, often in clusters

    • Clathrin coated pits → vesicles (endosomes)

    • Lysosomes are the usual recipient of the vesicles, where their hydrolytic activity acts on the endocytosed contents

  • Low pH is essential for enzyme activity—achieved by H+-ATPase activity.

Fig. 1.39 Formation of lysosome by receptor-mediated endocytosis.

Fig. 1.39 Formation of lysosome by receptor-mediated endocytosis.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p20 Oxford University Press.

Transcytosis (pinocytosis)

  • Way for macromolecules to cross cells (especially endothelial cells). Compounds are taken up in vesicles on one side, cross the cell in the vesicle, and are released by exocytosis on the other.

Cell cycle

Many cells in the body divide to replace cells that are lost through maturation and apoptosis or to respond to increased work imposed on a tissue. Multiple processes need to be carried out before a cell can divide:

  • Replication of chromosomes

  • Segregation of chromosomes into two diploid sets

  • Division of cytoplasm and cell membrane.

These processes have many potential problems because they involve large-scale replication and segregation of the genome. The cell cycle (Fig. 1.40) is a useful way of controlling the process of cell division into sequential steps which can be policed to ensure integrity of the cell progeny. There are several important checkpoints during the cycle which ensure that all necessary actions have been performed before progression to the next stage (e.g. that all chromosomes have duplicated before mitosis). These are detailed in the descriptions of the phases. These checkpoints are all controlled by the levels of checkpoint-specific cyclin-dependent kinase proteins.

Fig. 1.40 Schematic diagram of the cell cycle.

Fig. 1.40 Schematic diagram of the cell cycle.

Phases of the cell cycle

G1 (gap 1) phase

  • Variable length ⸫ major determinant of overall length of cell cycle

  • Most important phase for growth ⸫ high metabolic requirement

  • Contains the restriction point = cellular ‘decision’ as to whether to progress to S phase, and thus irreversibly to cell division, or to enter resting phase (G0).

S (synthesis) phase

  • Phase of DNA replication.

G2 (gap 2) phase

  • Phase of chromosome packaging

  • Synthesis of proteins required for mitosis

  • Checkpoint at the end of G2 to ensure that all DNA has been replicated before mitosis and that environmental conditions are favourable.

M (mitosis) phase

  • Actual physical division into two cells

  • Visible as mitotic figures in histological sections

  • Subdivided by the morphology of the chromosomes seen in histological sections (Fig. 1.41):

    • Prophase—chromosomes begin to condense to discretely visible structures

    • Metaphase—chromosomes line up on the equator of the nuclear spindle

    • Anaphase—chromosomes begin to pull apart into the two separate clusters

    • Telophase—chromosomes are now in the two tight clusters which will form the daughter cell nuclei

  • The nuclear spindle controls the segregation and movement of the chromosomes:

    • Spindle is composed of microtubules

    • Kinetochore attaches the centromere of each chromosome to the spindle

    • The spindle-attachment checkpoint only allows progression from metaphase to anaphase when all the chromosomes are attached to the nuclear spindle

    • Drugs such as vinblastine and colchicine destabilize the nuclear spindle microtubules and arrest mitosis, sometimes for days.

Fig. 1.41 Diagrams of the subprocesses within the M (mitotic) phase of the cell cycle.

Fig. 1.41 Diagrams of the subprocesses within the M (mitotic) phase of the cell cycle.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p24 Oxford University Press.

G0 (gap nought) phase

  • Not strictly part of the cell cycle but the resting state outside the cell cycle

  • Some cells remain in G0 until they die through senescence

  • Other cells remain in G0 for a variable length of time before re-entering the cell cycle.

Cell cycle control in human disease and disease treatment


  • Loss of cell-cycle checkpoint control points (= mutator phenotype) → accumulation of DNA damage and disarray → development of further malignant characteristics

  • Increased throughput through cell cycle → increase in number of neoplastic cells in relation to normal cells → overgrowth of neoplastic cells.


(Cellular structure and function OHCM10 p.657.)

  • Proliferation of smooth muscle cells in atherosclerotic plaques → increased vascular stenosis

  • Therapies are aimed at slowing or stopping cell cycle in these cells at time of angioplasty or stent placement to prevent restenosis. (e.g. Sirolimus-coated stents)

Alzheimer’s disease

(Cellular structure and function OHCM10 p.488.)

  • May be due to re-entry of quiescent neuronal cells into the cell cycle with abortive attempt at DNA replication → cell death by apoptosis after failure to pass checkpoint control.


(Cellular structure and function OHCM10 p.526.)

  • Induces DNA damage so entry into mitosis through the checkpoint at the end of G2 will either be delayed, while the DNA is repaired, or permanently prevented with apoptosis of the cell, if the DNA damage is irreparable

  • This is one of the mechanisms of therapeutic radiotherapy for the treatment of cancer.

Drug therapy of cancers

(Cellular structure and function OHCM10 p.524.)

  • Destabilization of the microtubules of the nuclear spindle (e.g. vinblastine, colchicine)

  • DNA damage, e.g. alkylating agents, such as chlorambucil, which covalently bind to DNA by alkyl groups

  • Drugs which bind to topoisomerase II (e.g. doxorubicin) which prevent this protein from functioning as the cleavage complex during DNA replication → prevention of entry to mitosis at the checkpoint at the end of G2.

Cell growth

The term ‘cell growth’ is used rather misleadingly in many texts, since strictly it refers to increase in size of cell without division, but it is usually used to mean increase in size of a tissue or organ by cell division. The cell cycle is the basic mechanism by which proliferation occurs, but it requires regulation by other factors, including growth factors and growth inhibitors.

Growth factors may arrive at a cell by three different routes:

  • Autocrine—when the factor is produced by the cell itself but acts back on it to stimulate growth. This may sound odd, and a positive feedback loop could lead to overproliferation, but it is an indicator of the metabolic well-being of the cell, which suggests that there is ‘room’ for more similar cells

  • Paracrine—when the factor is produced by a cell in close proximity to the cell it is affecting, mediated by short-range soluble molecules

  • Endocrine—when the factor is produced at some distance to the affected cells and carried to it by the blood. For example, thyroid-stimulating hormone (TSH), produced by the pituitary, which stimulates the growth of thyroid epithelial cells and the production of thyroxine.

Growth factors

There are a huge number of identified growth factors with generally long and complicated names that suggest where they were first discovered (e.g. vascular endothelial growth factor, transforming growth factor β‎). These names are confusing and often specifically named growth factors have a proliferative action on many different tissues.

The mechanism of action of these growth factors has a generic pattern:

  • The growth factor binds to a specific transmembrane cell surface receptor

  • The cell surface receptor either has an intrinsic enzyme (usually kinase) activity on its intracellular domain or is linked to a second messenger molecule which has such an activity

  • This sets off a signal transduction chain to the nucleus, where there is activation of transcription regulation factors → the transcription of more proteins → cells to pass into and through the cell cycle.

The specifics of each pathway are complex: it is enough to know if therapeutic intervention is planned in that specific area.

Growth inhibitory factors

In addition to positive growth factors, there are also a series of counterbalancing inhibitors of growth. These act by similar mechanisms to growth factors but result in the increased transcription of genes which code for inhibitors of the cell cycle, such as p27.



Genetically regulated form of cell death affecting individual cells. Distinguish from necrosis, which is the death of many adjacent cells due to some factor extrinsic to them (e.g. ischaemia). Apoptosis is derived from the Greek word meaning ‘dropping off’. Importantly, unlike necrosis, apoptosis is not pro-inflammatory.


  • Membrane blebbing

  • Cell shrinkage

  • Condensation of chromatin

  • Fragmentation of DNA

  • Expression of apoptotic markers on the cell surface to mark for phagocytic clearance

  • Phagocytosis by macrophages.

Initiators of apoptosis

  • Deprivation of survival factors (e.g. interleukin (IL)-1)

  • Proapoptotic cytokines (e.g. Fas, tumour necrosis factor)

  • Irradiation—gamma and ultraviolet

  • Anti-cancer drugs.

Intracellular regulators of apoptosis

  • bcl-2—suppresses the apoptotic pathway

  • p53.

Effectors of apoptosis

Caspases—a family of enzymes that cleave proteins close to aspartate residues. They have specific intracellular targets such as proteins of the nuclear lamina and cytoskeleton.

Physiological roles of apoptosis

  • Growth and development—loss of redundant tissue during organ development, e.g. interdigital webs, the majority of human neuronal cells produced during development also die during development

  • Control of cell number in adult life—regulation of balance between proliferation and cell death.

Role of apoptosis in disease

  • Increased apoptosis—acquired immune deficiency syndrome (AIDS), neurodegenerative diseases, post-ischaemic injury, hepatitis, graft-versus-host disease

  • Decreased apoptosis—many malignancies, autoimmune disorders (e.g. systemic lupus erythematosus).

Medical therapies which modulate apoptosis

  • Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs)—protect against colorectal adenomas and cancer by inducing apoptosis of colorectal epithelium through inhibition of COX-2

  • Anticancer drugs and radiotherapy (Cellular structure and function OHCM10 pp.524, 526)—induce apoptosis in tumour and normal tissues; both p53-dependent and independent mechanisms are recognized.


Humans are all developed from single cells, but the adult body contains about 250 specific cell types arranged into a large multiorgan system. The process by which a single progenitor cell produces these millions of specialized cells is called differentiation. The overall pattern of organization within a cell, which is synonymous with differentiation, is often referred to as its phenotype. Phenotype can be described at many different levels from its appearances by light microscopy, through to the pattern of proteins in its cytoplasm defined by mass spectrometry. The phenotype is often contrasted with the genotype of a cell (the description of its genetic material) but this boundary is becoming increasingly blurred as more and more interactions are being discovered between the genome and the rest of the cell.


The change in a cell from an undifferentiated to differentiated phenotype can be visualized by light microscopy and by electron microscopy (for tissue-specific organelles). Different types of epithelia are easily distinguished, but other specialized cells, especially lymphocytes, may require immunohistochemistry of cell surface proteins since their basic morphology is similar for different types (e.g. B vs T lymphocytes).

Genetic mechanism of differentiation

The process of differentiation is controlled by the regulation of the expression of genes. The detail of the mechanism for specific cell types has yet to be fully worked out but it will involve the known methods of gene regulation including promoters, repressors, and DNA methylation.


This is the change of a cell from one fully differentiated phenotype to a different, fully differentiated phenotype. It must be distinguished from dysplasia, which is a change to a less differentiated phenotype and is a precursor of cancer. Metaplasia occurs in a number of important sites in the human body:

  • In the distal oesophagus, there may be a change from squamous to glandular epithelium if there is gastro-oesophageal reflux, which exposes the squamous epithelium to the acidic contents of the stomach. The metaplasia results in Barrett’s oesophagus (Cellular structure and function OHCM10 p.695)

  • In the bronchi, there is often a metaplasia from ciliated glandular epithelium to squamous epithelium in cigarette smokers due to the irritant effect of the smoke. This results in the loss of the mucociliary escalator, the mechanism by which inhaled and secreted debris is removed from the bronchi, which leads to the development of chronic bronchitis. This metaplasia may precede dysplasia of the metaplastic squamous epithelium and the development of cancer

  • In the uterine cervix at puberty, the influence of hormones causes outgrowth of the cervix so that glandular endocervical epithelium is exposed to the vaginal environment, which stimulates metaplasia to squamous epithelium.

Meiosis and gametogenesis

The normal adult cell contains two copies of each chromosome (one inherited from the mother, the other from the father). In order to produce an oocyte or sperm, this needs to be reduced to a single copy. Thus, a special type of cell division is required (Fig. 1.42), which is called meiosis (from the Greek word meaning diminution). However, if this division simply segregated the chromosomes into pairs and put one of each pair into a cell that would become a gamete, then this gamete would contain individual chromosomes that came wholly from the mother or father. Since chromosomes contain many thousands of genes, this whole group of genes would always be passed on together as a single unit of inheritance. This would thwart evolutionary selection of the genome. Thus, within the process of meiosis there is a phase of recombination between maternal and paternal chromosomes before division, which leads to a much more thorough mixing of the genomes.

Fig. 1.42 The process of genetic recombination and segregation during meiosis.

Fig. 1.42 The process of genetic recombination and segregation during meiosis.

Reproduced with permission from Pocock G, Richards CD (2009). The Human Body 3e, p25 Oxford University Press.

Division I of meiosis

  • Each chromosome replicates to produce two sister chromatids which are tightly linked along their length

  • Each duplicated chromosome pairs with the equivalent chromosome from the other parent (the homologous chromosome) to form a structure called a bivalent. The sex chromosomes also participate in this process, even if male, because there is homology between some regions of the X and Y chromosomes

  • There is genetic exchange between the homologous chromosomes by crossing over of segments of the chromosomes (genetic recombination)

  • The bivalents line up on the mitotic spindle

  • Division leads to two cells each with a sister chromatid, with mixtures of maternal and paternal genes (i.e. haploid cell but with diploid amounts of genetic material).

Division II of meiosis

  • This is a simple segregation to divide the sister chromatids into genuine haploid cells.

Things that can go wrong during meiosis

  • Non-disjunction—in some divisions, a bivalent (in meiosis division I) or a sister chromatid (in meiosis division II) may not separate and so one cell will end up with no copies of that chromosome and another will have three. This occurs in around 10% of meiotic divisions. The embryos that develop from such cells are usually non-viable and account for the majority of spontaneous first-trimester abortions (‘miscarriages’) but some are viable, with recognized phenotypes (e.g. trisomy 21, producing Down syndrome)

  • Translocation, deletion, etc.—any of these can occur during the meiotic process and the crossing over of chromosomes makes this a more frequent occurrence in meiosis compared with mitosis.

Receptors and ligands

Pharmacology is the study of drugs and their actions. In order to fully understand how drugs work, it is first necessary to grasp some basic concepts of the chemical and physical principles that underlie drug–receptor interactions, as well as some of the nomenclature.


Cell surface receptors are proteins that are designed to recognize and respond to specific transmitter molecules, often known as ligands. The active site of a receptor is shaped and charged in such a way as to facilitate binding of its specific ligand, just as the active site of an enzyme is specific to its substrate.

Receptors can be crudely divided into two categories:

  • Those linked to ion channels

  • Those linked to G-proteins (Cellular structure and function p.[link]).

NB: receptors are not only found on the cell surface; enzymes and carrier proteins in the cytoplasm can also act as ‘receptors’ in the sense that their function is modified by binding of specific ligands. Furthermore, receptors that modulate gene expression are also found in or around the nucleus.

Irrespective of the protein receptor target, binding of a specific ligand instigates a chain of events that ultimately results in modification of cellular function (Cellular structure and function p.[link]).


Ligand is a general term used to describe any molecule that binds to a specific receptor. Endogenous ligands range from very simple inorganic molecules (e.g. nitric oxide (NO)) to complex organic molecules, including amino acids (e.g. glutamate), peptides, and lipid derivatives. Drugs are exogenous ligands that either mimic (agonists) or block (antagonists) the effects of their natural counterparts.

Ligand binding—general principles

Binding of a ligand (A) to a specific receptor (R) is a chemical interaction that results in the formation of a ligand–receptor complex (AR), often represented by the following equation:


A number of important chemical principles apply to this equation:

  • Law of mass action—the rate of reaction is proportional to the concentration of the reactants (Fig. 1.43a)

  • Equilibrium—when the rate of formation of AR equals the rate of dissociation of AR to A + R

  • The proportion of A that is complexed at equilibrium is determined by its affinity for the receptor, characterized by the affinity constant, k+1. The dissociation constant (k–1) is inversely proportional to k+1

  • k–1/k+1 is known as the equilibrium constant (KA) and is characteristic for a particular drug for a given receptor. KA equals the amount of drug required to occupy 50% of the receptor population at equilibrium

  • The proportion of receptors occupied at equilibrium is therefore dependent on both the equilibrium constant and the concentration of ligand present and is described by the Hill–Langmuir equation (Fig. 1.43b):

Fig. 1.43 Ligand binding. (a) Law of mass action. (b) The relationship between agonist concentration and receptor occupancy for an idealized agonist. This relationship is specific for a given drug and receptors in a particular tissue.

Fig. 1.43 Ligand binding. (a) Law of mass action. (b) The relationship between agonist concentration and receptor occupancy for an idealized agonist. This relationship is specific for a given drug and receptors in a particular tissue.

PA = [A][A]+KA

where PA = the proportion of receptors occupied at equilibrium.

Concentration–response relationship

Although receptor occupancy is an important consideration for pharmacologists, it is the response that a given concentration of ligand evokes in the body, or in an isolated piece of tissue, that is ultimately of greatest interest when exploring drug action.

At first glance, one might assume that ligand concentration would be related to response in the same way that it is to receptor occupancy. Theoretically, this is true for a ligand that evokes a maximum response when all the receptors are occupied. However, there are several factors that ensure that this is rarely the case in reality:

  • Availability—the ligand concentration delivered does not necessarily reflect the concentration at the receptors. This is especially true in vivo, when drug absorption and distribution, sequestration by plasma components, and breakdown by enzymes have a large impact on the concentration reaching the target receptors (Cellular structure and function p.[link])

  • Efficacy—the size of the response obtained for a given concentration of ligand is determined by the efficiency with which the second messenger system is evoked by its binding to the receptor. It may be the case, therefore, that binding of different ligands to an equal number of the same population of receptors produces different sized responses. Efficacy is the term given to describe the ability of a ligand to cause a physiological or cellular response and determines whether a ligand is an agonist (has efficacy) or antagonist (no efficacy)

  • A full agonist will cause a maximal response on binding all, or sometimes only a proportion of the receptors (the remainder is known as the receptor reserve) (see A and B in Fig. 1.44)

  • A partial agonist has lower efficacy than a full agonist and is unable to cause a maximum response, even when the concentration of agonist is sufficiently high to have 100% receptor occupancy (see C in Fig. 1.44)

  • An antagonist has very little or no efficacy. This means that binding of an antagonist to a receptor population fails to evoke any response, even when receptor occupancy is 100%.

Fig. 1.44 Log concentration response curves for idealized examples of full (A and B) and partial (C) agonists.

Fig. 1.44 Log concentration response curves for idealized examples of full (A and B) and partial (C) agonists.

NB: it is important to recognize that efficacy is unrelated to affinity. Thus, an antagonist can have high affinity (binds avidly to receptors) but zero efficacy (fails to produce a response).

Potency is a complex, non-specific pharmacological term sometimes misused to describe affinity on the basis of a drug’s ability to evoke a physiological response. As highlighted previously, response amplitude is not a good measure of affinity and the term can be misleading. That said, equipotent molar ratio (EPMR) is sometimes a useful measure to establish the relative amount of a given drug that is required to generate the same size of response as another drug in the same tissue (Fig. 1.45). The EPMR will be independent of the chosen response size (between ~25% and 75% of maximum) providing the concentration response curves are parallel. It should not be used in cases where slopes vary.

Fig. 1.45 Equipotent molar ratios (EPMRs) for drugs A, B, and C.

Fig. 1.45 Equipotent molar ratios (EPMRs) for drugs A, B, and C.

The EC50 of a drug is the effective concentration required to cause 50% maximal response for that drug (see Fig. 1.44). This value is related to agonist affinity but does not provide any information about efficacy. ED50 is the equivalent measure when using doses (e.g. mg/kg) instead of concentration (e.g. µM). IC50 is a measure to describe the concentration of an inhibitor required to cause 50% inhibition of a response.

The pD2 is –log of the EC50 (molar concentration). Thus, the pD2 for a drug with an EC50 of 1µM (10–6 M; see A in Fig. 1.44) is 6. The higher the pD2, the less drug is required to produce a 50% response.


Antagonists fall into several different classes, depending on their mode of action.

Competitive antagonists

  • Have affinity for the binding site of a specific receptor

  • Compete with the endogenous agonist for that site: the higher the affinity of the antagonist for the receptor, the lower the concentration that will be required to compete effectively with the agonist for receptors and reduce agonist receptor occupancy

  • The inhibitory effect of a given concentration of a competitive antagonist can be overcome by increasing the concentration of agonist and is, therefore, said to be reversible (Fig. 1.46)

  • A truly competitive antagonist must cause a parallel shift in concentration response curve without affecting the maximum response (Fig. 1.46).

Fig. 1.46 Competitive antagonism and dose ratio (DR) for several concentrations (1–100nM) of an idealized competitive antagonist.

Fig. 1.46 Competitive antagonism and dose ratio (DR) for several concentrations (1–100nM) of an idealized competitive antagonist.

The dissociation constant (KD) of a competitive antagonist can be established from experiments using an established agonist in the presence of different antagonist concentrations to determine the dose ratio (DR)—the ratio of agonist required to generate a given response in the presence of a known antagonist concentration compared to that in the absence of antagonist. Using the Schild equation or the Arunlakshana and Schild plot (Fig. 1.47), the KD (sometimes also referred to as the KB) can be determined. The KD for a true competitive antagonist is independent of antagonist concentration and, therefore, the gradient of the plotted line is equal to 1.0 (Fig. 1.47).

Fig. 1.47 Schild equation and plot to establish the dissociation constant (KD) for the competitive antagonist (B) shown in Fig. 1.46.

Fig. 1.47 Schild equation and plot to establish the dissociation constant (KD) for the competitive antagonist (B) shown in Fig. 1.46.

Irreversible competitive antagonists

  • Bind irreversibly but competitively to the agonist binding site, preventing agonist binding

  • Cause inhibitory effects that cannot be reversed by increasing the agonist concentration

  • Reduce the maximum response that can be achieved with an agonist

  • The irreversible nature of the effect means that the antagonistic action develops with exposure time, as an increasing number of receptors become irreversibly bound.

Non-competitive antagonists

  • Bind to a region of the receptor (or associated ion channel) other than the binding site for the endogenous agonist. Non-competitive antagonist binding can either alter the conformation of the agonist binding site to reduce the affinity for agonists, or prevent activation of the transduction mechanism required to evoke a response, or block ion channel opening

  • The effects cannot be reversed by increasing the agonist concentration and, therefore, the inhibitory action is reflected in a reduction in maximum response (Fig. 1.48).

Fig. 1.48 Effect of an irreversible (non-competitive) antagonist on the actions of an idealized agonist (A).

Fig. 1.48 Effect of an irreversible (non-competitive) antagonist on the actions of an idealized agonist (A).

Physiological antagonists

Agonists that cause the reversal of a physiological response via a different receptor and second messenger system (e.g. an agent that causes relaxation of a contracted muscle).

Chemical antagonists

React with the agonist before it binds to a receptor, either reducing the affinity of the agonist for the receptor or completely preventing its ability to bind.

Administration of drugs

In order for a drug to have a physiological effect in vivo, it must first be delivered in such a way that the correct amount of drug reaches the relevant receptors in the target tissue.

The chemical characteristics of the drug will determine:

  • How well it is absorbed into the bloodstream

  • How it will be distributed in the body

  • How quickly it will be metabolized and excreted from the body.

These are essential criteria in determining the chemical composition, dose, and frequency of dosing required to make for a useful pharmaceutical agent.

A number of routes are available for drug delivery. The route of administration is ultimately dependent on the chemical properties of the drug, but can usually be tailored for the particular application. For example, the most effective means of delivering a therapeutic agent to an asthmatic patient is directly to the bronchial tree by means of an inhaler or nebulizer. However, a cream would be the best form of drug delivery for a patient with eczema, eye drops for an eye infection, while a pill is most suitable in the treatment of stomach ulcers.

Intravenous delivery (injection or infusion)

  • The fastest means of delivery into the bloodstream

  • Avoids some of the problems associated with oral administration

  • Equally applicable in conscious and unconscious patients

  • Patients in hospital can receive chronic drug treatments via pump-driven or drip infusions

  • Generally unsuitable for self-administration.

Subcutaneous (under the skin) and intramuscular injections

  • Less rapid in onset than intravenous administration

  • Dependent on drug diffusion at the site of injection as well as local blood flow—an important consideration in patients in ‘shock’, where perfusion pressure is greatly reduced because of blood loss or vascular collapse

  • Generally, only implemented by medical staff, but patients with diabetes are often trained to inject themselves with insulin subcutaneously.

Intrathecal delivery

  • Used to deliver drugs directly into to the CSF

  • Lumbar puncture injections are technically challenging

  • Necessary for central nervous system (CNS) drugs that are unable to cross the blood–brain barrier, or that have undesirable side effects elsewhere in the body if injected systemically.

Delivery via the gastrointestinal tract (oral and rectal)

  • Oral administration is the most convenient because it does not necessarily require special equipment or medical supervision

  • Carries the disadvantage that the drug will be exposed to peptic enzymes and very acidic conditions in the stomach

  • Drugs that are not absorbed in the stomach will encounter different enzymes and alkaline conditions in the lower GI tract. Extreme changes in pH can lead to chemical changes that inactivate compounds and alter the rate at which they diffuse through cell membranes. For this reason, some agents (e.g. peptides like insulin that will be broken down by pepsin in the stomach) do not lend themselves to oral delivery. In such instances, alternative delivery routes or protective capsules can be used

  • Not appropriate in patients before operations and in those who are unable to swallow or are vomiting. In some instances, drugs can be delivered rectally instead

  • The rate at which orally ingested drugs are absorbed varies greatly in different patients and with different types of drug. Although some (e.g. aspirin, alcohol) are absorbed in the stomach, most are absorbed in the small intestine: gut motility is therefore a factor that determines the latent period between ingestion and the drug reaching its site of absorption. For this reason, taking an oral drug just after a meal is likely to slow its absorption by delaying its progress through the intestine. Once there, the rate at which it diffuses across the epithelium is determined primarily by its physico-chemical properties (e.g. particle surface area and equilibrium constant). Finally, the rate at which it enters the bloodstream is determined by the blood flow to the gut (splanchnic blood flow); rapid blood flow constantly removes drug from the gut wall and maintains the concentration gradient for diffusion. In general, however, peak absorption is usually achieved after ~1h and most of the drug has been absorbed within 4h.

Locally acting preparations

Where the desired site of action of a drug is accessible, it is desirable to deliver it directly to the target area to minimize (but not necessarily eliminate) systemic effects. The following are some examples of locally acting drug preparations:

  • Eye drops

  • Ear drops

  • Dermatological creams

  • Inhaled preparations for bronchial complaints.

Absorption, distribution, and clearance of drugs

Drug absorption

Drug absorption involves the passage of drug molecules across the epithelial barrier layer (e.g. intestinal epithelium, lung epithelium). The cells that constitute the barrier are tightly connected to one another, so the only means of passage is across the cell membrane (lipid bilayer). The most important means of diffusion of drugs is through the lipid membrane itself, requiring drug molecules to dissolve in both lipid and aqueous (water) environments.

Partition coefficient

The partition coefficient is a measure of the relative solubility of a compound in aqueous and lipid environments and is a critical determinant of drug absorption.


One of the key factors in determining the lipid solubility of a molecule is its readiness to ionize, generating a charged species that is repelled by uncharged lipid molecules. Most drugs can be categorized as acids or bases and, in general, the weaker the acid or base (i.e. less readily ionized), the greater the lipid solubility of the drug and the more rapid the diffusion across membranes.

pH partition

Acids and bases ionize as follows:

Acid:  AH  Ka A+  H+Base:  BH + Ka B+  H+

where Ka is the equilibrium constant for ionization.

Because drugs are generally weak acids and bases, the equilibrium is heavily weighted towards the non-ionized form, when the pH of the environment is neutral (~7.0).

Ionization is, however, affected by the pH of the environment in which the drug is dissolved:

  • A weak acidic drug ionizes more readily under alkaline conditions (pH >7)

  • A weak basic drug ionizes more readily under acidic conditions (pH <7).

The dissociation constant (pKa) is a term which allows for the environmental pH and is derived using the Henderson–Hasselbalch equation:

Weak acid: pKa = pH + log10 [AH]A-;  Weak base:  pKa = pH + log10 [BH+][B]

Remembering that drugs generally only cross membranes when they are not ionized, orally administered acidic drugs will be absorbed primarily in the stomach (pH ~3), while oral basic drugs will be absorbed in the small intestine (pH ~9).

Ion trapping

The rate of diffusion of a non-ionized molecule through a membrane is determined by the concentration gradient for that molecule across the membrane (Cellular structure and function p.[link]). As molecules diffuse down the concentration gradient, their concentration will tend to equalize between the two compartments. At equilibrium, the net diffusion of molecules will be zero because a concentration gradient no longer exists.

In reality, most drugs show a degree of ionization, as determined by the pKa of the drug and the pH of the compartment in which it is dissolved. Nevertheless, the non-ionized form of the drug will equilibrate across a membrane (Fig. 1.49). If the pH is the same in both compartments, the concentration of drug at equilibrium will be the same on both sides of the membrane. This is how drugs diffuse from cell to cell throughout a tissue.

Fig. 1.49 Ion trapping for a weak acid in the body compartment in preference to the acidic stomach compartment.

Fig. 1.49 Ion trapping for a weak acid in the body compartment in preference to the acidic stomach compartment.

However, the situation is more complicated when the pH is different between the two compartments. For example, a weakly acidic drug such as aspirin (pKa = 3.5) is absorbed in the stomach (pH ~3), where it passes first into the epithelial barrier layer and then into the blood (both of which pH ~7.4). The pKa for aspirin determines that it is mainly in the non-ionized form at this low pH, and can therefore readily diffuse across the epithelial cell membrane. Once inside the cell, however, the higher pH will cause a large proportion of the drug to ionize, rendering it unable to diffuse back through the membrane into the stomach compartment (Fig. 1.49). As a result, when the non-ionized form of the drug reaches equilibrium between the two compartments (stomach and intracellular), a large amount of drug is effectively ‘trapped’ within the cell in the ionic form.

Concentration and amount

When considering drug distribution between compartments, the relative size of the compartments is another important factor. At equilibrium, the concentration (i.e. amount per unit volume) (e.g. moles L–1, mg mL–1) of non-ionized molecules is the same in each of the compartments. However, by definition, the amount of drug (e.g. moles or mg) will only be the same if the volume of the compartments is the same. In reality, the volume of the compartments might be very different (Fig. 1.50). At equilibrium, under these conditions, although the concentration is the same in the compartments, there is considerably more of the drug in the body compartment. This is an important aspect in volume of distribution.

Fig. 1.50 The relationship between concentration and amount.

Fig. 1.50 The relationship between concentration and amount.

Drug distribution and clearance

Unless drugs can be applied directly to the target tissue (e.g. topical application for skin conditions, inhalation for asthma), we ultimately rely on the blood to deliver the drug to the relevant tissue. The amount of drug that reaches the tissue is dependent on absorption kinetics, blood flow to and from the target tissue, the rate of de-activation by metabolic processes, and subsequent excretion of the drug—collectively termed elimination.

Elimination of the vast majority of drugs occurs according to first-order kinetics. This is a term that describes the rate at which the plasma concentration of a drug falls over time. The characteristics of first-order kinetics are that, while the rate of drug elimination is dependent on its concentration, the time it takes for half of the drug to be eliminated is constant (the half-life). Thus, a plot of concentration against time falls exponentially on a linear scale (Fig. 1.51) but is linear on a semi-logarithmic scale. The half-life of a drug is a critical tool used by pharmacologists to predict plasma concentrations of a drug and to establish the interval required between dosing to maintain its therapeutic effects (i.e. to maintain a level above the effective concentration (Fig. 1.51)).

Fig. 1.51 An example of the plasma concentration profile for a single oral drug dose.

Fig. 1.51 An example of the plasma concentration profile for a single oral drug dose.

Dosing, steady state, and loading doses

A sound understanding of the pharmacokinetics of a particular drug is essential for determination of a suitable dosing regimen. For example, a drug with a very short plasma half-life will need to be administered frequently to maintain a therapeutic dose, while a bigger dose of a slowly absorbed drug might have to be administered to attain a therapeutic effect. It is a feature of drugs with first-order elimination kinetics that repeated doses at consistent intervals will ultimately result in generation of ‘steady-state conditions’, where the inter-dosing plasma concentration fluctuates between consistent maximum and minimum levels (Fig. 1.52). Ideally, a dosing strategy can be derived such that the plasma concentration rises rapidly to the therapeutic range and is maintained within the range by subsequent doses.

Fig. 1.52 An example of the plasma concentration profile for repeated oral drug dosing (1h intervals) of a drug with a half-life of ~30min. Note how repeated doses progressively increase the duration of the effective periods until steady state is reached.

Fig. 1.52 An example of the plasma concentration profile for repeated oral drug dosing (1h intervals) of a drug with a half-life of ~30min. Note how repeated doses progressively increase the duration of the effective periods until steady state is reached.

Zero-order kinetics: alcohol and salicylate

Alcohol and salicylate (the metabolite of aspirin) belong to a small group of compounds for which the rate of clearance is independent of its concentration (zero order). A plot of plasma alcohol concentration against time on a linear scale gives a straight line and the half-life is not a constant, as it is for most drugs (Fig. 1.53). The reason for the zero-order (or saturation) kinetics of these compounds is that the enzymes required to metabolize the drugs become saturated at very low levels; the enzyme is unable to increase its rate of activity in the face of increasing drug concentrations. Drugs that fall into this category are difficult to administer effectively because they do not attain steady state conditions.

Volume of distribution

The plasma concentration of a drug in the body is not only dependent on the amount of drug administered and the rate at which it is absorbed and eliminated, but is also dependent on the volume in which the drug is diluted. The plasma concentration of a drug that is confined to the bloodstream will accurately reflect the amount of drug in the body (Fig. 1.54a). However, the plasma concentration of a different drug, which is heavily absorbed in the aqueous component of tissue (Fig. 1.54b) and/or body fat (Fig. 1.54c), will vastly underestimate the amount of drug in the body because only a small fraction of the total drug is found in the blood. The apparent volume of distribution of a drug is calculated using the amount of drug administered and the concentration of the drug measured in the blood; by estimating the expected blood concentration if the drug distributes evenly throughout the water compartments of the body (i.e. blood, extracellular fluid, cellular fluid), we can determine whether the drug accumulates in the blood or distributes to other, non-aqueous compartments (e.g. subcutaneous fat; Fig. 1.54).

Desensitization, tachyphylaxis, tolerance, and drug resistance

Continual stimulation of receptors leads to the use of metabolically expensive second messengers. As a means of conservation, many receptor-mediated processes have in-built mechanisms to gradually damp down the effects of activation, → a time-dependent loss of cellular effects, sometimes known as tachyphylaxis. Tolerance is a term often used to describe a gradual loss of the desired physiological effects of a drug during the administration period. Drug resistance is a specific term used primarily for the loss of effect of anti-tumour drugs. Loss of agonist effect can be due to a number of reasons:

  • Receptor desensitization:

    • Usually rapid and caused by phosphorylation of G-proteins or conformational change in ion channels

    • Reversed by receptor removal and replacement with newly synthesized receptor

  • Adaptive loss of receptors:

    • A cellular response to continuous stimulation

    • Reduces drug efficacy

    • Can be reversed by removal of stimulus

  • Exhaustion of second messengers or essential metabolites:

    • Usually reversed during a period without stimulus

  • Physiological adaptation:

    • Relevant when homeostasis is upset by drug administration (e.g. a rapid drug-induced reduction in blood pressure is often compensated for by increased heart rate or blood volume).

Drug interactions

Before a drug can be prescribed, it is important to establish whether it might interact with any other medications that a patient is taking (Cellular structure and function OHCM10 p.757).

Interactions can take many forms:

  • Antagonistic: drugs can act to cancel each other out as a result of physiological, competitive, or non-competitive antagonism

  • Additive: drugs cause the same physiological effect and the total amplitude is the sum of the effects of the individual drugs

  • Synergistic: drugs cause the same physiological effect but the total amplitude of the response is considerably greater than the sum of the effects of the individual drugs. This can be related to a secondary interaction of drugs at the metabolic level, resulting in increased duration of action. Alcohol can have this effect because its metabolism depletes substances in the liver that are essential for metabolism of other drugs, slowing their clearance and prolonging their activity.

Indications, contraindications, and side effects

  • Indications are the clinical conditions for which a drug is prescribed

  • Contraindications are clinical conditions or other considerations (e.g. patient age, pregnancy) that preclude the use of a particular drug. For example, β‎-blockers (Cellular structure and function OHCM10 p.114), which are often prescribed for hypertension should not be given to patients with asthma as they can exacerbate airway constriction, and aspirin should not be prescribed to children under 12 years or to breastfeeding mothers. Furthermore, many drugs are unsuitable for patients with liver or kidney impairment because they are not cleared rapidly enough, → toxic effects

  • Side effects: unfortunately, very few drugs (if any) are so specific that they only cause the desired effect; most have secondary (off-target) effects (side effects) that might be undesirable and are responsible for the contraindications. As mentioned previously, β‎-blockers have the side effect of inhibiting β‎-adrenoceptors in the lung, where they can exacerbate bronchoconstriction in asthmatic patients and counteract β‎-adrenoceptor agonists prescribed to help alleviate asthma.


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