9780521516358book.pdf

17.3.2 Experimental procedures for ligand-binding studies

The general experimental approach for studying the kinetics of receptor–ligand

binding and hence to determine the experimental values of the binding constants

and the total number of binding sites is to incubate the receptor preparation with the

ligand under defined conditions of temperature, pH and ionic concentration for a

specific period of time that is sufficient to allow equilibrium to be attained. The

importance of allowing the system to reach equilibrium cannot be overstated as

equations 17.1 to 17.11 do not hold if equilibrium has not been attained. Using an

appropriate analytical procedure, the bound and unbound forms of the ligand are then

quantified or some associated change measured. This quantification may necessitate

the separation of the bound and unbound fractions. The study is then repeated for a

Table 17.2Receptor preparations for the study of receptor–ligand

binding

Receptor preparation Comments

Tissue slices 5–50mm thick, generally adhered to gelatine-coated glass slide.

Good for study of receptor distribution.

Cell membrane

preparation

Disrupt cells (from tissue or cultures) by sonication and isolate

membrane fraction by centrifugation. Lack of cytoplasmic

components may compromise receptor function. Used to study

ligand binding and receptor distribution in lipid rafts and

caveolae. Increasingly commonly used with cell lines

transfected with human receptor genes.

Solubilised receptor

preparation

Disrupt membrane with detergents and purify receptors by

affinity chromatography using an immobilised competitive

antagonist. Isolation from other membrane components may

compromise studies.

Isolated cells Release cells from tissue by mechanical or enzymatic

(collagenase, trypsin) means. Cells may be in suspension or

monolayers. May be complicated by presence of several cell

types. Widely used for the study of a range of receptor functions.

Allows ligand binding and cellular functional responses to be

studied under the same experimental conditions.

Cultured cell lines Very popular. Has advantage of cell homogeneity and ease of

replication.

Recombinant receptors Produced by cloning or mutagenesis techniques and inserted into

specific cultured cell line including ones of human origin. Popular

for the study of the effect of mutations on receptor function such

as constitutive activity (CAMs) and cell signalling. Care needed

to ensure that receptors have same functional characteristics

(e.g. post-translational modification) as native cells.

681 17.3 Ligand-binding and cell-signalling studies