Guanosine diphosphate binding,metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes

Nucleotides such as GTP and GDP appear to be involved in signal transduction via G protein modulation of adenylate cyclase activity. Studies on direct binding of ³H]GDP to membranes prepared from cultured immature rat Sertoli cells indicated that this process was reversible, approached steady state within 10 min, had a K_a of 4.5 ·10⁶M⁻¹ and was specific for guanine nucleotides. The non-hydrolyzable analog, guanosine 5′-O-3-thio]triphosphate (GPPPS]), was most effective as an inhibitor of ³H]GDP binding (ED₅₀ = 4.8·10⁻⁸M), whereas guanosine 5′-O-2-thio]diphosphate (GppS]) was less potent (ED₅₀ = 3.4·10⁻⁷M). Release of bound GDP was enhanced by follitropin (FSH) in the presence of GpppS], although not by FSH alone. Sertoli cell membranes possess guanine nucleotide hydrolase activity, where 95% of added nucleotide was rapidly degraded to guanosine. Binding kinetics were significantly influenced by nucleotide metabolism, which was prevented by controlling the Mg²⁺ concentration with EDTA and including AppNH]p to reduce nonspecific hydrolysis. Kinetic studies indicated that GppS] inhibited (P < 0.05) GpppS]-stimulated adenylate cyclase activity (K_i = 1.8·10⁻⁷M), whereas basal activity remained unaffected. Addition of GppS] to pre-activated enzyme (FSH plus GTP) resulted in a time-dependent decay of adenylate cyclase activity with a K_off value of 6 ± 1·min⁻¹. Using a two-stage pre-inculbation technique, adenylate cyclase activity was demonstrated to be sensitive to the nucleotide bound. When FSH was included, catalytic activity was not altered by the order of pre-incubation with the nucleotides. This suggested that the exchange of bound GppS] for GpppS] was enhance by FSH. Activation and attenuation of FSH-sensitive adenylate cyclase activity is dependent on a nucleotide exchange mechanism which is driven by (1) the higher affinity of G for GTP than GDP, (2) enhanced release of GD when FSH is present and (3) GTP hydrolysis coupled to rapid metabolism of guanine nucleotides.