Abstract
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.
Highlights
The interaction of forskolin with adenylate cyclase inotropic effect on the heart and tolower blood pressure [1]
The adenylate cyclase of bovine sperm, which is with protective effects of calmodulin.Calmodulin has insensitive to activation by forskolin, was not protected been suggestedto activatedirectly the catalytic unoitf a brain by forskolin against inactivation by either N-ethylmal- adenylate cyclase [9],by a mechanism independent of a fully eimide or heat
The studies reported here demonstratea protective effect of forskolin against inactivation of adenylate cyclase by exposure to heat or toN-ethylmaleimide. Such proexcess MnC12, 100 ~ L fMorskolin increased the apparentK,for tection implies fist, that forskolin-adenylate cyclase interac
Summary
EFFECTS ON SUBSTRATE KINETICS AND PROTECTION AGAINST INACTIVATION BY HEAT AND N-ETHYLMALEIMIDE*. Consesitive adenylate cyclase from brain and the particulate quently, Seamon and Daly ( 5 ) suggested that stimulation of enzyme from platelets were inactivated upon preincu- adenylate cyclase by forskolin may be a direct effect of forbation with N-ethylmaleimide. Inthisreport,the interactions of forskolin with the catalytic unit have been further examined by subjecting theenzyme to such denaturation conditions and by evaluating its effects on the kinetic of the adenylate cyclases from brain, platelets, eryth- behavior of the enzyme withrespect to metal and metal-ATP. Half-maximal activation of the platelet functional guanine nucleotide regulatory component, and iits adenylate cyclase was seen with 3 to 10 ~ C fMorskolin, known that calmodulin protects this cyclase against thermal and the Kd for forskolin, determined from heat inacti- inactivation [9, 10].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
