Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex

Abstract

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose and 2 heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5''-guanylyl imidodiphosphate and NaF. The MW of the protein component was estimated as 328000 with a smaller form of MW 153000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150000, 47000 and 35000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave 1 product of 170000 daltons on SDS gels. The catalytic subunit of the calmodulin-sensitive enzyme apparently is 150,000 .+-. 10,000 daltons and that the enzyme exists as a complex of 1 catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a MW of 150,000 .+-. 10,000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., and Storm, D.R.].