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
What is the advantage of using a scratchard plot?
the plot is linear so it is easier to determine the Kd and Bmax
therefore you get a relatively accurate Bmax without having to use saturating concentrations of radioligand
also provides visual indication of whether or not binding is a simple biomolecular process or a more complex process involving heterogeneous population of receptors
Does the presence or absence of GTP or its non-hydrolysable analogue GTP-gammaS affect the Kd for agonist radioligands binding to GPCR?
Agonists have a higher affinity for the coupled receptor compared to the uncoupled
Resting state, the heterotrimer binds GDP but its affinity is reduced by its interaction with the activated receptor- GDP is consequently replaced by GTP
Heterotrimeric G protein dissociates when GTP binds to alpha subunit, while alpha subunit binds GTP it remains dissociated - usually GTP is hydrolysed by the alpha subunit to give GDP which enables association to beta-gamma subunit and therefore enable heterotrimer to reform
GTP-gamma S cannot be hydrolysed by the alpha subunit of the heterotrimeric G protein so alpha subunit cant recombine with the beta-gamma subunit so the receptor cannot bind to the G protein so receptor remains in the uncoupled state therefore reducing affinity for agonist
What is a good example of cooperative binding ?
interaction of oxygen with haemoglobin
haemoglobin reversibly binds 4 oxygen molecules but the binding of each molecule is not independent of each other
1st binds with low affinity but this facilitates the binding of the others
What does positive cooperativity result in?
it results in an increase in binding affinity
- induces upward concave curve in a scratchard plot
What does negative cooperativity result in?
it results in a decrease in affinity
- induces a downward convex curve in scratchard plot
What does a Hill plot show?
It is a graphical method showing cooperativity
What are the axis’ of a Hill plot ?
x-axis= log free concentration y-axis= log (B/Bmax-B)
How is the Kd calculated from the Hill plot and what is the slope ?
X-intercept= nlogKd
slope (n)= number of ligand molecules bound by the receptor
What is indirect binding ?
also known as competition binding
it is the binding of unlabelled ligands to a receptor by displacing a suitable radioligand from the macromolecule
it is useful when only a few radioligands are available for a receptor type
What happens in a competition binding assay ?
the concentration of unlabelled ligand is increased and the effects this has on specific binding of a fixed concentration of radioligand is studied, enabling the IC50 to be calculated
What is the IC50?
this is the concentration of unlabelled ligand that inhibits the specific binding of the radioligand by 50%
- it can be used to estimate Ki
What is the Ki ?
it is the equilibrium dissociation constant for the unlabelled ligand - to do this you use the cheng and prusoff equation: IC50/(1+ [L*]/Kd)
When is indirect binding a useful technique?
When screening large numbers of compounds for binding a particular receptor
