The Vasopressin-sensitive Adenylate Cyclase of the Rat Renal Medulla

A partially purified plasma membrane fraction was prepared from the rat renal medulla by flotation in a discontinuous sucrose density gradient. The preparation was enriched in vasopressin-stimulated adenylate cyclase activity to a greater extent than either fluoride-stimulated or basal activity. The Km for vasopressin was 4 to 15 x 10-9 m in different preparations. The Km for ATP was 0.12 to 0.16 mm for basal, hormone-stimulated, and fluoride-stimulated activities. The different reaction rates under the three conditions were due to differences in the maximal velocity of the reaction. The concentration of Mg++ required for half-maximal enzyme activity was the same for basal, vasopressin-stimulated, and fluoride-stimulated activities. The activation energy of the reaction was 15 to 17 Cal per mole for all three conditions. The reversibility of hormone stimulation of adenylate cyclase was studied by measuring the rate of change of enzyme activity following dilution of the hormone. Decreasing the concentration of hormone led to an immediate drop in the rate of formation of adenosine cyclic 3':5'-monophosphate to that expected at the lower vasopressin concentration. This indicates that the hormone is in equilibrium with the receptors and that the binding of hormone is rapidly reversible.