Oxidation of critical cysteine residues of type I adenylyl cyclase by o-iodosobenzoate or nitric oxide reversibly inhibits stimulation by calcium and calmodulin.
The calmodulin binding domain of the type I adenylyl cyclase has recently been identified as an amino acid sequence (residues 495-522) that contains 2 cysteine residues. Therefore, we examined the effect of several sulfhydryl reagents on the calmodulin sensitivity of the enzyme. Treatment of membranes containing the type I adenylyl cyclase with N-ethylmaleimide rapidly inhibited basal, calcium/calmodulin-stimulated, and forskolin-stimulated adenylyl cyclase activity. When the enzyme was treated with limiting amounts of o-iodosobenzoate, which oxidizes vicinal sulfhydryls to disulfides, stimulation by Ca2+ and calmodulin was eliminated at concentrations which did not affect basal adenylyl cyclase activity. Calmodulin stimulation of the enzyme was restored by treatment with dithiothreitol or glutathione which reduce disulfides to free thiols. NO and sodium nitroprusside also reversible inhibited calmodulin stimulation of the enzyme. We propose that the loss in calmodulin sensitivity caused by these reagents may be due to the oxidation one or more sets of vicinal thiols present in the enzyme.
Duhe
R J
RJ
Department of Pharmacology, University of Washington, Seattle 98195.
Nielsen
M D
MD
Dittman
A H
AH
Villacres
E C
EC
Choi
E J
EJ
Storm
D R
DR
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation; D005033:Ethylmaleimide / Q000494:pharmacology; D007463:Iodobenzoates / Q000737:chemistry*; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry*; D009599:Nitroprusside / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000067956:Adenylyl Cyclase Inhibitors; D002147:Calmodulin; D007463:Iodobenzoates; D013438:Sulfhydryl Compounds; D009599:Nitroprusside; C008154:2-iodosobenzoic acid; D009569:Nitric Oxide; D000262:Adenylyl Cyclases; D003545:Cysteine; D005033:Ethylmaleimide; D002118:Calcium