ligand binding

Question 1

What was used prior to radioligand binding assays ?

Answer 1

Use of biological responses of a receptor to its endogenous ligands and drugs

Question 2

What are the disadvantages of measurements by biological response?

Answer 2

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

Question 3

What are the advantages of radioligand binding assays?

Answer 3

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

Question 4

What is the equation used to explain radioligand binding ?

Answer 4

[L] + [R] = [RL]

• K1= forward reaction
• K2= reverse reaction

Question 5

What do K1 and K2 indicate?

Answer 5

They are constants which remain unchanged

They describe the velocity of the forward and backward reactions

Question 6

What is Kd?

Answer 6

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

Question 7

What is K ?

Answer 7

it is the association constant
K= 1/Kd
K= [RL]/[R][L]

Question 8

What are the units of Kd?

Answer 8

M - concentration

Question 9

What is Bmax?

Answer 9

it is the total concentration of receptors

= [R] + [RL*]

Question 10

What does this equation indicate, [RL]=(Bmax[L])/([L*]+Kd)?

Answer 10

it predicts that the relationship between the concentration of receptor-ligand complex and concentration of free ligand is hyperbolic and saturable

Question 11

What is a decrease in Bmax associated with ?

Answer 11

A down regulation of a receptor population

Question 12

What is an increase in Bmax associated with ?

Answer 12

An up regulation of a receptor population

Question 13

What are the similarities between the ligand binding isotherm and a michaelis plot of enzyme velocity and substrate concentration?

Answer 13

both are hyperbolic
Bmax= Vmax
Kd= Km

Question 14

What is the most common method for separating bound from free ligand?

Answer 14

Filtration - receptor/macromolecules are trapped on the filter that is freely permeable to the free ligand

Question 15

How is filtration carried out?

Answer 15

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

Question 16

What is non-specific binding ?

Answer 16

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

Question 17

How does non-specific binding increase?

Answer 17

it increases in a linear manner as free ligand concentration increases

Question 18

How is non-specific binding measured?

Answer 18

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

Question 19

How to calculate non-specific binding ?

Answer 19

it can be measured by subtracting it from total binding

Question 20

what is total binding ?

Answer 20

specific binding + non-specific binding

Question 21

What is a limitation of ligand binding ?

Answer 21

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

Question 22

Why can the total ligand concentration be assumed to be effectively the free ligand concentration?

Answer 22

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

Question 23

What is a scratchard plot ?

Answer 23

it is a useful method for analysing binding data
y-axis= ratio of bound to free ligand
x-axis= bound ligand concentration

Question 24

what is the slope of a scratched plot equal to and what is the intercept on the x-axis?

Answer 24

slope= -1/Kd 
x-intercept= Bmax

Question 25

What is the advantage of using a scratchard plot?

Answer 25

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

Question 26

Does the presence or absence of GTP or its non-hydrolysable analogue GTP-gammaS affect the Kd for agonist radioligands binding to GPCR?

Answer 26

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

Question 27

What is a good example of cooperative binding ?

Answer 27

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

Question 28

What does positive cooperativity result in?

Answer 28

it results in an increase in binding affinity

- induces upward concave curve in a scratchard plot

Question 29

What does negative cooperativity result in?

Answer 29

it results in a decrease in affinity

- induces a downward convex curve in scratchard plot

Question 30

What does a Hill plot show?

Answer 30

It is a graphical method showing cooperativity

Question 31

What are the axis’ of a Hill plot ?

Answer 31

x-axis= log free concentration 
y-axis= log (B/Bmax-B)

Question 32

How is the Kd calculated from the Hill plot and what is the slope ?

Answer 32

X-intercept= nlogKd

slope (n)= number of ligand molecules bound by the receptor

Question 33

What is indirect binding ?

Answer 33

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

Question 34

What happens in a competition binding assay ?

Answer 34

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

Question 35

What is the IC50?

Answer 35

this is the concentration of unlabelled ligand that inhibits the specific binding of the radioligand by 50%
- it can be used to estimate Ki

Question 36

What is the Ki ?

Answer 36

it is the equilibrium dissociation constant for the unlabelled ligand - to do this you use the cheng and prusoff equation: IC50/(1+ [L*]/Kd)

Question 37

When is indirect binding a useful technique?

Answer 37

When screening large numbers of compounds for binding a particular receptor