ligand binding Flashcards
What was used prior to radioligand binding assays ?
Use of biological responses of a receptor to its endogenous ligands and drugs
What are the disadvantages of measurements by biological response?
In vivo- lipid solubility, metabolism and excretion and attenuation of responses due to feedback mechanisms limit the study of receptor function
Presence of spare receptors, uptake mechanisms, partial agonism and desensitisation complicate the interpretation of their response
Difficult with classical techniques to study relationship between receptor structure and its function and between drug structure and receptor binding
What are the advantages of radioligand binding assays?
Used to estimate number or concentration of receptors in a tissue- therefore it is used to investigate the distribution of receptors amongst tissues and effect of disease or drug intervention upon their distribution
Used to discriminate between multiple receptor types and estimate relative proportions within tissues
Investigate the relationship between receptor structure and function
What is the equation used to explain radioligand binding ?
[L] + [R] = [RL]
- K1= forward reaction
- K2= reverse reaction
What do K1 and K2 indicate?
They are constants which remain unchanged
They describe the velocity of the forward and backward reactions
What is Kd?
It is the equilibrium dissociation constant and [R], [L] and [RL] are the concentrations of free receptor, free ligand and ligand receptor complex
Kd= K2/K1
What is K ?
it is the association constant
K= 1/Kd
K= [RL]/[R][L]
What are the units of Kd?
M - concentration
What is Bmax?
it is the total concentration of receptors
= [R] + [RL*]
What does this equation indicate, [RL]=(Bmax[L])/([L*]+Kd)?
it predicts that the relationship between the concentration of receptor-ligand complex and concentration of free ligand is hyperbolic and saturable
What is a decrease in Bmax associated with ?
A down regulation of a receptor population
What is an increase in Bmax associated with ?
An up regulation of a receptor population
What are the similarities between the ligand binding isotherm and a michaelis plot of enzyme velocity and substrate concentration?
both are hyperbolic
Bmax= Vmax
Kd= Km
What is the most common method for separating bound from free ligand?
Filtration - receptor/macromolecules are trapped on the filter that is freely permeable to the free ligand
How is filtration carried out?
it is done at 4 degrees to minimise dissociation of the ligand from the receptor and the filter paper is assayed in a liquid scintillation counter to determine amount of ligand bound
What is non-specific binding ?
it is the binding of the ligand to anything other than the receptor such as the assay tubes, an array of membrane or cell macromolecules
- it is unsaturable
How does non-specific binding increase?
it increases in a linear manner as free ligand concentration increases
How is non-specific binding measured?
it is measured in the presence of a high concentration of a unlabelled competing ligand
the competing ligand displaces L* from the receptor complex so only radioligand binding is present at non-specific sites
How to calculate non-specific binding ?
it can be measured by subtracting it from total binding
what is total binding ?
specific binding + non-specific binding
What is a limitation of ligand binding ?
the ligand must be radio labelled and due to cost of radioligands it means normally the max concentration is well below that required for saturation of the receptor
Why can the total ligand concentration be assumed to be effectively the free ligand concentration?
because normally the bound ligand concentration is very small relative to the total concentration of ligand
This means it is common to find binding plotted against total ligand concentration rather than the free ligand concentration which is difficult to measure using filtration to separate bound from free
What is a scratchard plot ?
it is a useful method for analysing binding data
y-axis= ratio of bound to free ligand
x-axis= bound ligand concentration
what is the slope of a scratched plot equal to and what is the intercept on the x-axis?
slope= -1/Kd x-intercept= Bmax