Hormone-induced guanyl nucleotide binding and activation of adenylate cyclase in the Leydig cell.

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Abstract
The adenylate cyclase activity of [rat] Leydig cell homogenates and membrane fractions is highly dependent on guanyl nucleotides, and enzyme responses to luteinizing hormone [lutropin] or human chorionic gonadotropin are small in the absence of guanyl nucleotides. In the presence of 10 .mu.M guanosine 5''-[.beta.,.gamma.-imido]triphosphate Gpp[NH]p, both hormones consistently stimulated testicular adenylate cyclase activity by up to 200%. Leydig cell membranes bound [3H]Gpp[NH]p at 30.degree. C with high affinity (Ka = 1.5 .times. 107 M-1) and binding capacity of 60 pmol/mg of protein. During kinetic studies, the association rate constant was 1.7 .times. 106 M-1 min-1, and the dissociation constant was 0.038 min-1. In the presence of gonadotropin (10 pM to 10 nM), concentration-dependent increases of 40-100% in Gpp[NH]p binding were observed in Leydig cell membranes. Kinetic studies showed that gonadotropin decreased the association rate constant to 0.73 .times. 106 M-1 min-1 and the dissociation rate constant to 0.017 min-1, with no effect on the equilibrium binding constant. Thus, the increase in Gpp[NH]p binding was not due to a change in receptor affinity but was attributable to increased availability of nucleotide binding sites. The 50% effective dose for adenylate cyclase activation by gonadotropin in the presence of Gpp[NH]p was identical with that observed for gonadotropin-induced binding of the GTP analog (50 nM). Gonadotropin-induced binding of Gpp[NH]p in Leydig cell membranes may represent interaction with the guanyl nucleotide regulatory site during hormonal activation of adenylate cyclase.

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