RECONSTITUTED ADENYLATE-CYCLASE FROM BOVINE BRAIN - FUNCTIONS OF THE SUBUNITS

Stimulation of soluble adenylate cyclase from bovine cerebral cortex by guanosine 5''-(.beta.,.gamma.-imino)triphosphate (Gpp(NH)p) requires the interaction of 2 enzyme components: the catalytic unit (C) and the guanine nucleotide regulatory unit (G/F). The G/F unit from bovine cerebral cortex can be detected by its ability to restore Gpp(NH)p-responsiveness to the brain catalytic unit prepared according to S. Strittmatter and E. J. Neer. The G/F unit can be solubilized with the nonionic detergent, Lubrol 12A9, or with cholate and can be separated from adenylate cyclase by gel filtration. Using separated components of adenylate cyclase, the sites of action of regulatory ligands were localized. The purine-specific site or adenosine inhibition and the site for activation by calmodulin are on the separated catalytic unit. The rate-limiting step in guanine nucleotide activation of adenylate cyclase can be defined more explicitly. The isolated G/F unit is activated by Gpp(NH)p with a half-time of 52 .+-. 2 min at 23.degree. C. Addition of 0.2 M (NH4)2SO4 decreases the half-time to 8 .+-. 3 min. The G/F unit can be stripped of endogenous nucleotides but this does not affect the rate of activation by Gpp(NH)p, nor the ability of (NH4)2SO4 to increase this rate. Release of tightly bound GDP from the G/F unit is not the rate-limiting step in the activation of the soluble enzyme. The rate-limiting step seems to be a slow conformational or structural change in the G/F unit. This change may be a dissociation of the G/F unit into an active form since (NH4)2SO4 (0.2 M) decreases the apparent MW of the G/F unit from 190,000 to 88,000. Hormones stimulate adenylate cyclase by increasing the rate of activation of the enzyme by guanine nucleotides. If the processes which occur in solution reflect those which occur in membranes, hormone receptors may act by promoting the dissociation of the G/F unit from an inactive to an active form.