The role of calcium in the control of adrenal adenylate cyclase. Enhancement of enzyme activation by guanyl-5'-yl imidodiphosphate.

Abstract

To define the role of Ca2+ and other divalent cations in the hormonal control of adrenal adenylate cyclase activity, we have examined adrenocorticotropic hormone (ACTH) binding, enzyme activation, and the catalytic formation of cAMP independently of one another.When ethylene glycol bis( fi-aminoethyl ether)N,N,N'tetraacetic acid (EGTA)-treated rat adrenal membranes were exposed to ACTH a t 4"C, rinsed, and resuspended in hormone-free buffer, the loss of functional hormone-receptor complexes was slow (half-life = 4 h).Ca2+, M&+, and Mn2+ had no detectable effects, either on the number of active complexes initially formed or on the decay rate of formed complexes.To measure the rate of enzyme activation, adrenal membranes pretreated with EGTA or EDTA were exposed at 23°C either to guanyl-5'-yl imidodiphosphate (Gpp(NH)p) alone or to Gpp(NH)p plus ACTH in the presence or absence of metal ions.caz+ (0.1 to 2 mM) caused a 3-to ?-fold increase in the rate of activation by both Gpp(NH)p alone and Gpp(NH)p plus hormone.Mg2+ and MnZ+ were much less effective than Ca2+ in promoting activation.In EDTA-pretreated rat liver membranes, M&+ was a more effective promoter than Ca2+, and pretreatment with EGTA did not impair the rate of Gpp(NH)p activation.To examine the effects of metal ions directly on the catalytic unit, preactivated adrenal enzyme was solubilized and isolated after chromatography.In the presence of a constant high concentration of M&+ (8 mM), catalytic activity was stimulated by MnZ+ and inhibited by CaZ+ in a competitive and dose-dependent manner.We conclude that the stimulatory effect of Ca2+ on ACTH action is exerted at a step involving guanine nucleotide interaction with the enzyme complex.Ca2+ can also act as a direct inhibitor of the enzyme by competing with Mn2+ €or interaction at a second site, probably located on the catalytic subunit.Calcium ion has long been known to play an important role in the control of steroidogenesis by ACTH' in the adrenal gland.Among the earliest events stimulated by ACTH is cyclic AMP generation, a process which is thought to govern the rate-limiting conversion of cholesterol into A5-pregnenolone (1) in the steroidogenic pathway.Calcium ion facilitates