OXIDATION OF CRITICAL CYSTEINE RESIDUES OF TYPE-I ADENYLYL-CYCLASE BYO-IODOSOBENZOATE OR NITRIC-OXIDE REVERSIBLY INHIBITS STIMULATION BY CALCIUM AND CALMODULIN

The calmodulin binding domain of the type I adenylyl cyclase has recen tly been identified as an amino acid sequence (residues 495-522) that contains 2 cysteine residues. Therefore, we examined the effect of sev eral sulfhydryl reagents on the calmodulin sensitivity of the enzyme. Treatment of membranes containing the type I adenylyl cyclase with N-e thylmaleimide rapidly inhibited basal, calcium/calmodulin-stimulated, and forskolin-stimulated adenylyl cyclase activity. When the enzyme wa s treated with limiting amounts of o-iodosobenzoate, which oxidizes vi cinal sulfhydryls to disulfides, stimulation by Ca2+ and calmodulin wa s eliminated at concentrations which did not affect basal adenylyl cyc lase activity. Calmodulin stimulation of the enzyme was restored by tr eatment with dithiothreitol or glutathione which reduce disulfides to free thiols. NO and sodium nitroprusside also reversible inhibited cal modulin stimulation of the enzyme. We propose that the loss in calmodu lin sensitivity caused by these reagents may be due to the oxidation o ne or more sets of vicinal thiols present in the enzyme.