Photoaffinity labeling of brain adenylate cyclase preparations with azido[125 I]iodocalmodulin.
A partially purified calmodulin-sensitive adenylate cyclase from bovine cerebral cortex was photoaffinity labeled with azido[125 I]iodocalmodulin. Sodium dodecyl sulfate gel electrophoresis followed by autoradiography revealed several cross-linked polypeptides ranging in molecular weight from 37000 to 200000. The calmodulin-sensitive enzyme was submitted to a number of purification steps to determine if any of the calmodulin binding polypeptides copurified with adenylate cyclase activity. Fractionation procedures used included Bio-Gel A5M and Ultragel AcA 34 gel chromatography, isoelectric focusing, and native gradient gel electrophoresis. One cross-linked peptide having a molecular weight of 170000 correlated with adenylate cyclase activity through all purification steps. Native gradient gel electrophoresis in the presence of 0.03% deoxycholate gave one peak of adenylate cyclase activity with a Stokes radius of 40 A, consistent with a molecular weight of 140000-150000. It is proposed that the molecular weight of the adenylate cyclase catalytic subunit is 150,000 and that each catalytic subunit interacts with one calmodulin.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D002850:Chromatography, Gel; D007457:Iodine Radioisotopes; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010782:Photolysis
D000345:Affinity Labels; D001386:Azides; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D007457:Iodine Radioisotopes; C038199:azidoiodocalmodulin; D008345:Manganese; D000262:Adenylyl Cyclases; D002118:Calcium