Relationship between forskolin and calcium-calmodulin stimulation of rat cerebral cortex adenylate cyclase: enzyme activation modulates substrate (MgATP) affinity

In membranes prepared from the rat cerebral cortex EGTA 0.4-100 mumol/l was found to dose-dependently inhibit adenylate cyclase activity, both during basal conditions and when the cyclase activity had been stimulated with 10 mumol/l forskolin. Addition of calcium 2-30 mumol/l (in excess of EGTA) totally prevented the inhibition induced by EGTA, both in the absence and presence of forskolin. These data are consistent with the notion that in the absence of EGTA the rat cortex adenylate cyclase is considerably stimulated by endogenous calcium-calmodulin. The data also show that stimulation with endogenous calcium-calmodulin is more than additive with that of forskolin indicating an action on identical cyclase units. The kinetics of the rat cortex adenylate cyclase for its substrate (MgATP) was also investigated. Activation of the cyclase with endogenous calcium-calmodulin, induced a marked increase in the Vmax of the enzyme and a concomitant almost 2-fold increase in the Km. Stimulation with 10 microM forskolin also induced a marked increase in the Vmax as well as an almost 3-fold increase in the Km. A combination of the two stimulants caused a further increase in Vmax as well as Km; the Vmax being increased more than 17-fold and the Km being increased 3.5-fold over the basal. The data indicates that both calcium-calmodulin and forskolin induces a similar type of activation of the adenylate cyclase which is associated with a lowered affinity for its substrate.