Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.
A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one 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 molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology*; D005576:Colforsin / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D012969:Sodium Fluoride / Q000494:pharmacology
D002147:Calmodulin; D046911:Macromolecular Substances; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D012969:Sodium Fluoride; D000262:Adenylyl Cyclases; D008274:Magnesium