Adrenal cortex adenylate cyclase

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Abstract
Speci fic binding sites for 5′-guanylyl-imidodiphosphate [Gpp(NH)p] have been identified in a partially purified plasma membrane fraction from bovine adrenal cortex. The apparent affinity of Gpp(NH)p at 30° C was 12 μM−1 and the concentration of binding sites was 100 pmoles per mg of protein. Binding of Gpp (NH)p is inhibited by Mn2+ > Mg2+ ≫ Ca2+ and enhanced by low concentrations of the chelators ethylenediamino-tetraacetic acid (EDTA) and ethylene glycolbis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA). High concentrations of EDTA are inhibitory and at 2.5 mM EDTA binding of Gpp(NH)p is only 10% of that observed in the absence of the chelator. The bound labeled GTP analogue exchanged only slowly with the unlabeled nucleotide after a steady state has been reached. EDTA also releases the bound labeled Gpp(NH)p from its binding sites. The slow dissociation of Gpp(NH)p can explain the persistent activation of adenylate cyclase observed after pretreatment of bovine adrenal cortex plasma membranes with Gpp(NH)p and subsequent washing. It is suggested that at least parts of these binding sites are identical to the sites identified earlier as regulatory sites for angiotensin high-affinity receptors (Glossmann et al., 1974a) and for ACTH-stimulated cyclase (Glossmann and Gips, 1974).