Resolution and properties of the catalytic unit of cardiac adenylate cyclase

Abstract

The catalytic unit (C) of cardiac adenylate cyclase was resolved from the guanine nucleotide-binding regulatory protein (G/F) by fractionation of Lubrol 12A9 extracts with 33% saturated (NH4)2SO4 and by gel filtration in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS). Catalytic activity in both preparations was supported by Mg2+ and Mn2+, Vmax values being 12 fold higher in the presence of the latter cation. NaF-activated preparations were also subjected to gel filtration in the presence of CHAPS. In this case, both the catalytic activity and G/F emerged in the activated state. G/F could be deactivated by dialysis of the preparation in the absence of NaF; the catalyst however remained activated following dialysis. NaF sensitivity was reconstituted in C by nonactivated and NaF-activated preparations of G/F isolated by gel filtration. Reconstitution was dependent upon the amount of both G/F and C in the assay. Nonactivated G/F also reconstituted guanine nucleotide sensitivity in C. Catalytic activity was thermally labile, but was stabilized at 25 degrees C by substrate (Mn2+ ATP). C was stimulated up to 25-fold by forskolin. The NaF-activated catalyst resolved by gel filtration was relatively insensitive to this agent. Forskolin, however, augmented NaF-sensitivity by both non-activated and NaF-activated G/F provided it was added to the assay before G/F. Similarly, forskolin augmented guanine nucleotide sensitivity of nonactivated G/F in the presence of GTP gamma S. The P site agent 2',5'-dideoxyadenosine (DDA) was a weak inhibitor of nonactivated C, but a powerful inhibitor of C stimulated by forskolin or activated by G/F in the presence of NaF. In all respects C precipitated by (NH4)2SO4 appeared to be identical with that resolved by gel filtration. Ammonium sulfate precipitation, because of its simplicity, speed, relatively good yield, and adaptability for large scale operation, may be the preferred method for preparing cardiac C for purification studies