Substance P: characteristics of binding to synaptic vesicles of rat brain

Abstract

1. The binding of substance P (SP) to synaptic vesicles from rat brain was studied by use of the 125I-Tyr8-analogue of SP. 2. The pH dependence of the binding of both peptides to the lipid extractable fraction of synaptic vesicles was shown to be comparable. 3. The binding of 125I-Tyr8-SP shows a rate constant of association (k1 = 6.6 x 10(6) M-1 S-1), a rate constant of dissociation (k-1 = 6.4 x 10(-4) S-1) and gives a KD of 1 x 10(-10) M. Kd derived from equilibrium studies was 3.2 x 10(-10) M. 4. The binding of 125I-Tyr8-SP to lipids of synaptic vesicles was shown to be reversible, saturable and highly specific. 5. The kinetic data suggest one population of binding sites with a maximal number of 0.8 pmol per mg protein of the synaptic vesicle preparation. 6. Unlabeled SP and the (2--11)-, (3--11)- and (4--11)-analogues of SP inhibit the binding of 125I-Tyr8-SP in a decreasing order in a competitive way when added in excess. Tyr8-SP and eledoisin did not interfere with the binding of 125I-Tyr8-SP whereas uperolein and neurotensin caused a partial inhibition. Physalaemin and D-Ala2-D-Met5-enkephalin enhance the binding of 125I-Tyr8-SP in a cooperative way.