Ligand Binding

Question 1

Disadvantages of studying biological responses (either in vivo or in vitro)

Answer 1

• In vivo; lipid solubility, metabolism, excretion and attenuation of responses by feedback mechanisms all limit the study of receptor function
• Presence of spare receptors, uptake mechanisms, partial agonism and desensitisation can complicate interpretation
• Difficulty in studying relationship between receptor structure and function and drug structure and receptor binding

Question 2

Advantages of radioligand binding assays

Answer 2

• Can estimate number/concentration of receptors on a tissue (used to investigate receptor distribution and see effect of disease/drug intervention on characteristics of receptor population)
• Used to discriminate between multiple receptor types; especially subtypes to estimate their relative proportions in a single tissue

Question 3

Does an increase or decrease in receptor population down regulate Bmax

Answer 3

Down regulation of receptors shows a decrease in Bmax

Question 4

Does an increase or decrease in receptor population up regulate Bmax

Answer 4

Up regulation of receptors shows an increase in Bmax

Question 5

Why would a curved scatchard plot occur?

Answer 5

Presence of a heterogenous receptor population or the receptor and receptor-effector complex have different affinities for the ligand

Question 6

Which region of a receptor is important for agonist binding?

Answer 6

Transmembrane region, usually a full response seen but lower agonist affinity

Question 7

Definition of Bmax

Answer 7

Total concentration of receptors in a sample of tissue

Question 8

Definition of Kd

Answer 8

Equilibrium dissociation constant, small Kd = stronger affinity, weaker Kd = weaker binding

Question 9

Process for separating bound ligand from free ligand?

Answer 9

Receptor and other macromolecules in which the ligand is bound are trapped on the filter paper, which is freely permeable to free ligand
Filtration is done rapidly at 4 degrees to minimise dissociation of the ligand from the receptor
Filter paper is then assayed using liquid scintillation counter to determine the amount of ligand bound

Question 10

Definition of non-specific binding

Answer 10

Any site that a ligand will interact with that is not the receptor site, ligand will interact loosely. Non-specific binding sites lack the key specific structural requirements for the ligand to bind

Question 11

Total ligand binding

Answer 11

Sum of the specific and non-specific binding

Question 12

Definition of specific binding

Answer 12

Ligand binding to its target receptor

Question 13

Limitation of ligand binding

Answer 13

Ligand must be radio-labelled, which gives a high cost and a lot needs to be used for saturation.
Total ligand concentration is effectively free land concentration

Question 14

Advantage of a scatchard plot

Answer 14

Linear, can accurately measure Bmax within needing saturation

Question 15

What is co-operative binding

Answer 15

Increasing binding increases affinity due to changes in structure

Question 16

How do you work out Kd from a hill plot?

Answer 16

X axis intercept, nlogKd

Question 17

What does the slope of a hill plot represent

Answer 17

n - number of ligand molecules bound to receptor

Question 18

What is indirect (competition) binding

Answer 18

Binding of unlabelled ligands to a receptor can be indirectly characterised by their ability to displace suitable radioligand from the macromolecule

Question 19

IC50 defintion

Answer 19

concentration of unlabelled ligand the inhibits specific binding of radiolabel by 50%