Purification of the regulatory component of adenylate cyclase.

Abstract

The regulatory component (G/F) of adenylate cyclase (EC 4.6.1.1) from rabbit liver plasma membranes was purified essentially to homogeneity. The purification was accomplished by 3 chromatographic procedures in sodium cholate-containing solutions, followed by 3 steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 .mu.mol.cntdot.min-1.cntdot.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 recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain 3 polypeptides with MW of .apprx. 52,000, .apprx. 45,000 and .apprx. 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 MW 52,000 and 45,000 polypeptides with 32P.