Interactions of forskolin and adenylate cyclase. Effects on substrate kinetics and protection against inactivation by heat and N-ethylmaleimide.

The interaction of forskolin with adenylate cyclase was studied by evaluating its effect on metal and metalATP kinetics and by measuring its protective effect when the enzyme was subjected to denaturation conditions. The solubilized calmodulinand forskolin-sensitive adenylate cyclase from brain and the particulate enzyme from platelets were inactivated upon preincubation with N-ethylmaleimide. Forskolin protected against this inactivation in a concentration-dependent manner and demonstrated Kd values of 6.3 and 7.6 PM for the brain and platelet adenylate cyclases, respectively. Protection against N-ethylmaleimide inactivation of the brain enzyme was also afforded by calmodulin, but not to the extent seen with forskolin. Forskolin also protected against thermal inactivation of the adenylate cyclases from brain, platelets, erythrocytes, and 549 lymphoma wild type and cycvariants. The adenylate cyclase of bovine sperm, which is insensitive to activation by forskolin, was not protected by forskolin against inactivation by either N-ethylmaleimide or heat. Half-maximal activation of the platelet adenylate cyclase was seen with 3 to 10 ~ C M forskolin, and the Kd for forskolin, determined from heat inactivation kinetics, was 9 to 11 PM. Activation of the platelet adenylate cyclase by forskolin was negatively cooperative (n = 0.59) with respect to forskolin. This activation occurred without change in Michaelis or dissociation constants for free M&’, but coincided with a &fold increase in the corresponding constants for MgATP and a 2-fold increase in the K , for MnATP. However, forskolin did not affect the K , app for MnATP of the solubilized adenylate cyclase from brain. These results imply binding of forskolin by adenylate cyclase. The data suggest that the same binding site for forskolin is involved in both protection and activation and that this binding site is distinct from those to which the substrates bind.