Products of reaction catalyzed by purified rat liver guanylate cyclase determined by 31p NMR spectroscopy.
Products of the reactions catalyzed by highly purified preparations of soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] from rat liver were identified and quantified with 31P NMR spectroscopy. Utilization of this technique necessitated modification of the standard assay conditions; higher concentrations of enzyme and substrate (2 mM), Mg2+ instead of Mn2+, and longer incubation times (up to 46 hr) at 30 degrees C were used. Revision of our reported procedure for purificaton of guanylate cyclase [Tsai, S-C., Manganiello, V. C. & Vaughan, M. (1978) J. Biol. Chem. 253, 8452-8457] to include chromatography on Ultrogel AcA34 and agarose-hexane-GTP provided an enzyme with specific activity higher than in our earlier preparations. 31P NMR spectra obtained during incubation of this enzyme showed that the rates of GTP disappearance and cyclic GMP (cGMP) accumulation were constant for approximately 16 hr. They indicated, however, that the preparations were contaminated with inorganic pyrophosphatase. This was removed by preparative electrophoresis, yielding enzyme with specific activities (900-1300 nmol/min per mg of protein) higher than those reported for guanylate cyclases from rat liver or lung. With this preparation, cGMP and PPi were the only products of GTP detected, consistent with the assumption that the guanylate and adenylate cyclase reactions are analogous.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D011756:Diphosphates / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010710:Phosphates / Q000378:metabolism; D051381:Rats
D011756:Diphosphates; D010710:Phosphates; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase