Receptor Theory Flashcards
how do you measure the amount of specific binding sites?
set up 2 sets of test tubes in parallel
1) radioactive ligand + tissue
2) radioactive ligand + tissue + cold ligand in excess
Majority of hot ligand will bind to the non-specific binding sites as they will be displaced by the cold ligand
What are the requirements of radioactive ligands?
1) radioactivity can’t be too strong (distort structure) or too weak (undetectable)
2) ligand must be biologically active, chemically pure
3) ligand must not be degraded= stored in free radical scavenger (ethanol), in the dark, low temperature and incorporation of anti-oxidant
What are the pros and cons of using 3H as a radiolabel
pros= 12.5 year half life, very stable, indistinguishable from native compound cons= require specialised lab and very expensive
What are the pros and cons of using 125I as a radiolabel
pros= cheap, highly specific if compound as aromatic OH (e.g. tyrosine), easy to make cons= very unstable, short half life, biological activity can be reduced
What are the tissue incubation conditions?
Need to maintain the integrity of the tissue and the ligand
1) low temperature
2) additives= e.g. antiproteases- protect ligand and tissue
3) tissue can be isolated membrane, cell culture or slices
4) Need protein conc of 0.1-1mg/ml
How do you separate bound ligand from free and what are the problems?
1) centrifugation= wash pellet due to non-specific binding
2) soluble binding= column chromatography, dialysis, precipitation and absorption
If the ligand has a low affinity= high Kd= there is a higher dissociation rate.
What is the Langmuir equation and what do each of the components mean?
It describes the relationship between receptor occupancy, affinity and #drug concentration. Kd is the measure of affinity at 50% occupancy and is a constant. If it differs it means that the receptor is different. High Kd means low affinity
What is the scatchard plot?
y= bound/free, x= bound
x intercept= Bmax
slope= -1/Kd
Is used to accurately measure Kd
