Receptor Theory I and II Flashcards
What is occupancy?
The proportion of receptors occupied varies with drug concentration
What is the equation for occupancy?
Occupancy = number of receptors occupied
total number of receptors
What do values of 0 and 1 represent in terms of occupancy?
0 = no drug present 1 = all receptors occupied
How can occupancy be measured?
A radioligand binding assay
Why can occupancy not be measured from response?
High occupancy will produce a larger response, however response is also effected by efficacy
Describe how a radioligand binding assay is carried out
- Prepare the protein sample (containing the receptors)
- Mix with the radioactive drug of interest
- Incubate
- Filter and rinse away unbound drug
- Measure the number of complexes
What is one of the issues with the radiolingand binding assay?
Ligands can non-specifically bind to tissue, glass etc
How can non-specific binding of ligands be reduced and what are some examples?
Anti-absorbants
Albumin or collagen for peptides
O-catechol for catecholamines
How experimentally can you discriminate between specific and non-specific binding?
Have two set of test tubes
Set one will contain tissue + radiolabelled ligand
Set two will contain tissue + radiolabelled ligand + unlabelled (cold) ligand
In set one there will be both specific and non-specific binding
In set two the unlabelled ligand will out compete the radiolabelled ligand for the recognition site so there will be no specific binding in this set
What are the conditions that a ligand must meet for the radioligand binding assay?
Must be extremely chemically pure
Labelling of the drug with radioactivity but reach very high specific activity to allow very low tracer concentrations
How is degradation of the ligand avoided?
Free radical scavenger (e.g. ethanol) in drug solution
Store at low temperature
Avoiding light
Incorporation of antioxidant (ascorbic acid)
What are the advantages and disadvantages of 3H as a ligand radio-label?
Advantages: Labelled product indistinguishable from its native compound High specific activity can be obtained Good stability when properly stored Long half-life (12.5 years) Disadvantages: Specialised labs required Expensive and difficult to label
What are the advantages and disadvantages of 125I as a ligand radio-label?
Advantages:
If the compound has an aromatic group it can be easily incorporated at high specific activities
Iodination easy and cheap
Disadvantages:
Readily degraded
Biological activity can be decreased over time
Short half life
How do you separate tissue + bound ligand from free ligand?
Centrifugation Filtration Dialysis Column chromatography Precipitation/adsorption
What is a problem when separating bound ligand from free ligand?
The rate of dissociation of the ligand-receptor complex
Speed of separation must be compatible with affinity of receptor
Lower affinity will require a quicker and more efficient method
