Specific Binding of [3H]Lysine-Vasopressin to Pig Kidney Plasma Membranes

Abstract

Abstract A plasma membrane fraction was prepared from pig kidney medulla. It contained lysine-vasopressin-sensitive adenylate cyclase activity (maximum activation: 4- to 6-fold, apparent Km value for the hormone: about 5 x 10-9 m). Lysine-vasopressin was not inactivated by the membrane preparation even at low hormonal concentrations. The specific [3H]lysine-vasopressin (vasopressin) binding sites found are probably involved in adenylate cyclase activation; binding occurred in a concentration range giving dose-dependent activation of the adenylate cyclase. Relative affinities of vasopressin, oxytocin, (O-methyl)-tyrosine2-oxytocin and angiotensin (vasopressin g oxytocin g (O-Me)-Tyr2-oxy g angiotensin) for binding and adenylate cyclase activation were similar. Nonspecific binding, i.e. binding which could not be inhibited by 10-5 m unlabeled vasopressin, represented 12.5% of the specific binding at 10-8 m [3H]vasopressin. For all hormonal concentrations, [3H]vasopressin binding increased with time up to an equilibrium value. The binding was reversible. The time course of hormone-receptor complex formation was highly temperature- and hormone concentration-dependent. Dissociation and association rate constants at 30° varied from 0.02 to 0.088 min-1 for k-1, and from 1.3 x 10+7 to 3.0 x 10+7 m-1 min-1 for k1. For the same enzyme preparation, the k-1:k1 ratio was very close to the value deduced from the hormonal concentration giving half the maximum binding at equilibrium. When adenylate cyclase activation was measured as a function of time after the addition of hormone, activation was found to be progressive. The time required to reach maximum activation is similar to that needed to reach an equilibrium value for [3H]vasopressin binding at the same concentration. These data suggest that adenylate cyclase activation is a function of receptor occupancy. Reversal of adenylate cyclase activation was also progressive. When adenylate cyclase activation at equilibrium was measured for different values of receptor occupancy, it was found that for the same increment in receptor occupancy, the increment in adenylate cyclase activation drops as total receptor occupancy increases.