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

Abstract

A partially purified calmodulin-sensitive adenylate cyclase from bovine cerebral cortex was photoaffinity labeled with azido[125]iodocalmodulin. Sodium dodecyl sulfate gel electrophoresis followed by autoradiography revealed several cross-linked polypeptides ranging in MW from 37,000 to 200,000. 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 MW of 170,000 correlated with adenylate cyclase activity through all purification steps. Native gradient gel electrophoresis in the presence of 0.03% deoxycholate gave 1 peak of adenylate cyclase activity with a Stokes radius of 40 .ANG., consistent with a MW of 140,000-150,000. The MW of the adenylate cyclase catalytic subunit is probably 150,000 and each catalytic subunit interacts with 1 calmodulin.