Photoaffinity labeling of brain adenylate cyclase preparations with azido[125I]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.