Kinetic analysis of inositol trisphosphate binding to pure inositol trisphosphate receptors using scintillation proximity assay.
Inositol 1,4,5-triphosphate (InsP3) receptors are regulated by many intracellular signals including proteins and small messengers. By linking purified cerebellar InsP3 receptors to scintillation proximity assay beads, binding of radioligands can be measured without separation of bound from free ligand. InsP3 receptors assayed by scintillation proximity assay bound heparin with high affinity and stereoselectively bound InsP3 with similar affinity to cerebellar membranes. By rapidly freezing scintillation proximity assay reactions and then counting the frozen samples, both fast and slow components of [3H] InsP3 association and dissociation were identified. Our novel freeze-quench method in combination with conventional stopped-quench equipment and scintillation proximity assay allows the rapid kinetics of the interactions of pure receptors with their ligands to be resolved.
Patel
S
S
Department of Pharmacology, University of Cambridge, United Kingdom.
Harris
A
A
O'Beirne
G
G
Cook
N D
ND
Taylor
C W
CW
D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D012588:Scintillation Counting / Q000379:methods*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate