The influence of purified, preactivated N, 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, protein increased the Vof 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,-protein-activated human platelet adenylate cyclase was inhibited by the hormone, epinephrine, and by the stable GTP analogs, guanosine 5'-[fi,y-imido]triphosphate and guanosine 5'-[y-thioltriphosphate. The stable GTP analog or hormone-induced adenylate cyclase inhibition was not competitive with regard to the concentration of N, protein. Inhibition of N,-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, and Ni, 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, and Ni, respectively.The hormone-sensitive adenylate cyclase [ATP pyrophosphate lyase (cyclizing)] is apparently regulated by two distinct guanine-nucleotide-binding regulatory sites. The guanine nucleotide site (N,) mediating stimulation of adenylate cyclase has been purified to apparent homogeneity from several tissues [I -31. This site is not only involved in hormonal stimulation of adenylate cyclase but also in enzyme stimulation by stable GTP analogs, cholera toxin and fluoride [41. In cyc-variants of S49 lymphoma cells, this site is at least partially missing [5, 61. Therefore, the adenylate cyclase of these cells is not stimulated by hormones, guanine nucleotides, fluoride or cholera toxin, but the system can be reconstituted by insertion of purified N, protein derived from other tissues [I -31. On the other hand, a separate guanine-nucleotide-binding regulatory site (N,) appears to be involved in adenylate cyclase inhibition by hormones and guanine nucleotides [7]. The evidence for this site has recently been corroborated by data obtained in the N,-deficient cyc-cells. In these cells, stable GTP analogs decrease basal adenylate cyclase activity and enzyme activity stimulated by the diterpene, forskolin [8,9]. Furthermore, the N,-deficient cyc-adenylate cyclase can be inhibited by the peptide hormone, somatostatin, in a GTP-dependent manner [lo], similarly as described for hormone-induced adenylate cyclase inhibition in complete cellular systems [I 1 -131. In the studies on cyc-adenylat...