Ca2+-dependent regulation of guinea pig brain adenylate cyclase.

The effect of Ca2+ on guinea pig bra in adenylate cyclase has been studied using metal buffers to regulate the free Ca2+ concentration. Particulate fractions prepared in the presence of CaZ+ (to bind all of the available calmodulin to all of the available binding sites) exhibited a biphasic response to Ca2+. The first was a 2fold stimulation of basal cyclase activity (half-maximal = 0.08 p ~ ) . At higher [Ca2+], the cyclase was inhibited to -80 to 90% of basal (half-maximal inhibition = 0.3 p ~ ) . Particulate fractions prepared by exhaustive washing with EDTA and ethylene glycol bis(P-aminoethyl ether)N,N,iV’,iV’-tetraacetic acid (EGTA) (in an att empt to deplete the membranes of calmodulin) exhibited no stimulation of adenylate cyclase by Ca2+ but did show the Ca2+-dependent inhibitory phase (halfmaximal inhibition = 0.5 p ~ ) . The calmodulin content of this preparation (EGTA-membranes) was 0.24 pg/mg of protein. The activation phase could be restored to EGTA-membranes by addition of calmodulin. The CaZ+ dependence of calmodulin-dependent phosphodiesterase exhibited a half-maximal activation at [Ca”] = 0.3 p ~ , similar to the half-maximal inhibition of cyclase in the two preparations. Likewise, 51.2’ activation of phosphodiesterase (half-maximal stimulation = 0.1 mM) occurred over the same [SI-*’] range as did half-maximal inhibition of cyclase (0.3 mM) in EGTAand Ca-membranes. The Ca2+ and Sr2+ dependence of calmodulin tyrosine fluorescence (an indirect measure of Ca2+ and Sr2+ binding) were essentially the same as the Ca2+ and Sr2+ dependence of phosphodiesterase activation and the inhibition of the cyclase. These results suggest that the Ca2+ dependent inhibition of adenylate cyclase may be mediated either by calmodulin or by a metal binding site which has binding properties similar to calmodulin. Thus, our results show that the re is an inverse relationship between adenylate cyclase activity and phosphodiesterase activity and suggest that intracellular calcium may mediate a coordinated regulation of the rates of synthesis and degradation of cyclic nucleotides.