Purification of the regulatory component of adenylate cyclase.
The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.
D000262:Adenylyl Cyclases / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011817:Rabbits
D046911:Macromolecular Substances; D009243:NAD; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000262:Adenylyl Cyclases; D005459:Fluorides