L6 - Receptor Theory II

Question 1

What is efficacy?

Answer 1

Full agonist will have an efficacy > 0
An antagonist will have an efficacy = 0
The maximum response achievable from an applied dose or agent.

Question 2

What is occupancy?

Answer 2

The proportion of receptors occupied will vary with drug concentration
No drug present occupancy = 0
All receptors occupied occupancy = 1

Question 3

In the case of agonist, the size of the response produced is proportional to?

Answer 3

Occupancy

This directly allows you to measure affinity

Question 4

Radioligand binding assays of a ligand to a protein target are used to?

Answer 4

Measure affinity
The ligand could be
- Neurotransmitter, hormone, growth factor, cytokine, drugs, toxins
The protein target could be
- Receptors, ion channels, enzymes, carrier molecules

Question 5

Radioligand binding assay steps

Answer 5

1. Prepare protein sample
2. Mix with drug on interest (drug needs to be made radioactive)
3. Incubate to allow reaction to reach equilibrium
4. Filter and rinse the drug away
5. Count and analyse data

Question 6

Non-specific binding

Answer 6

Most ligands bind non-specifically to tissue, filter paper, glass.
Measuring proportion of specific and non-specific binding is key
- Rinsing only removes unbound radioligand from incubation medium
Bound radioactivity = non specific + specific binding

Question 7

Non-specific binding to filters & glass can be reduced by?

Answer 7

Anti-absorbents
- Albumin or collagen for peptides
- O-catechol for catecholamines
But this does not reduce non-specific binding to the tissue under study

Question 8

Non-specific binding method

Answer 8

1. Set up 2 rows of test tubes with increasing concentrations of the radioactive drug
2. In one row add excess non-radioactive drug
3. These will out compete the radioactive drug for the specific binding site (because they are in excess

Question 9

The radioligand

Answer 9

The ligand must be biologically active since its binding to recognition site correlates with a response
The ligand must be extremely pure chemically
Labelling of the drug with radioactivity must achieve high specific activity to allow very low concentrations

Question 10

Ways to solve degradation

Answer 10

Free-radical scavenger in drug solution
Store at low temperature
Avoiding light
Incorporation of antioxidant (e.g. ascorbic acid)

Question 11

Advantages of 3H as a radio label

Answer 11

Labelled product indistinguishable from native compound
High specific activity
Good stability when properly stored
Long half life – 12 years

Question 12

Disadvantages of 3H as a radio label

Answer 12

Specialised labs required

Labelling is expensive and difficult

Question 13

Advantages of 125I as a radio label

Answer 13

If compound has an aromatic hydroxyl group can be incorporated at high specific activities
Iodination easy and cheap in most labs

Question 14

Disadvantages of 125I as a radiolabel

Answer 14

More readily degraded
Biological activity of ligand can be reduced
Short half life – 67 days

Question 15

Tissue and incubation conditions - tissue types

Answer 15

Isolated membranes
Slices
Synaptosomes
Cultured cells 
Solubilized/purified receptors

Question 16

Role of incubation

Answer 16

Must preserve integrity of both ligands and binding site
Additives used to protect tissue/ligand
- Protease inhibitors for peptides
- Antioxidants for ligand

Question 17

Incubation conditions

Answer 17

Temperature usually around 0oC

Membrane protein concentration in range of 0.1-1 mg/ml with assay volumes 0.25-1 ml

Question 18

Separating bound from free techniques

Answer 18

Filtration or centrifugation
For soluble binding 
- Dialysis
- Column chromatography
- Precipitation/adsorption

Question 19

Issues with separating bound from free

Answer 19

Major problem is rate of dissociation of ligand-receptor complex
Speed of separation must be compatible with affinity of ligand for receptor
Lower affinity (higher KD) requires faster and more efficient separation

Question 20

Scatchard plot non-specific binding line

Answer 20

Linear- not on log
Non-linear when on log
Radioactive ligand and cold excess
Not saturable

Question 21

Scatchard plot

Answer 21

Specific binding = total bound - nonspecific binding
Data is usually plotted on a semi-log scale
Radioligand binding is stereospecific

Question 22

Scatchard plot total bound line

Answer 22

Increases rapidly then slows down
Radioactive ligand
Saturable because total number of receptors is limited

Question 23

Scatchard plot specific binding line

Answer 23

Increases rapidly then plateaus (sigmoidal)

There will be a concentration of ligand where all the receptors will be labelled and the curve will plateau

Question 24

The Scatchard equation

Answer 24

B/F = (Bmax- B)/KD

Question 25

Langmuir equation

Answer 25

Describes the relationship between receptor occupancy, affinity and drug concentration
Langmuir equation/data is converted to a scatchard plot

Question 26

Scatchard plot analysis - F

Answer 26

Free ligand = concentration of ligand added (mol/litre)

Question 27

Scatchard plot analysis - B

Answer 27

Measured specific binding

Question 28

Scatchard plot analysis - Bmax

Answer 28

(pmol/mg protein)
Maximum amount of drug which can bind specifically to the receptors
If one molecule binds to each receptor, then it indicates the concentration of receptors

Question 29

Scatchard plot analysis - Kd

Answer 29

(mol/litre)
The dissociation constant for the radioligand determined by saturation analysis
The concentration of ligand which, at equilibrium, occupies 50% of the receptors

Question 30

Low Kd

Answer 30

High affinity

Question 31

High Kd

Answer 31

Low affinity