Inhibition of Ns‐Protein‐Stimulated Human‐Platelet Adenylate Cyclase by Epinephrine and Stable GTP Analogs

Abstract

The influence of purified, preactivated N_s protein, mediating adenylate cyclase stimulation by hormones and guanine nucleotides, was studied on adenylate cyclase activity in membranes of human platelets. The preactivated coupling protein caused a large increase in platelet enzyme activity. The activation occurred after a short lag phase and exhibited saturation. N_s protein increased the V of the platelet adenylate cyclase without change in the enzyme's affinity for the substrate, MgATP, whereas the apparent affinity for Mg²⁺ was increased by more than one order of magnitude. The N_s‐protein‐activated human platelet adenylate cyclase was inhibited by the hormone, epinephrine, and by the stable GTP analogs, guanosine 5′‐[β,γ‐imido]triphosphate and guanosine 5′‐[γ‐thio]triphosphate. The stable GTP analog or hormone‐induced adenylate cyclase inhibition was not competitive with regard to the concentration of N_s protein. Inhibition of N_s‐protein‐stimulated platelet adenylate cyclase caused by stable GTP analogs appeared to be persistent, was amplified by epinephrine and was accompanied by a large decrease in the enzyme's apparent affinity for Mg²⁺. The data suggest that the hormone‐sensitive adenylate cyclase is regulated by two guanine‐nucleotide‐binding sites, N_s and N_i, mediating enzyme stimulation and inhibition, respectively, by hormones and guanine nucleotides, and that the two coupling components interact in a non‐competitive manner with the adenylate cyclase, exhibiting high and low affinity for Mg²⁺ when affected by N_s and N_i, respectively.