title: Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside.
abstract: Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D005292:Ferricyanides / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D006427:Hemin / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D051381:Rats; D012966:Sodium Cyanide / Q000494:pharmacology
chemical_list: D005292:Ferricyanides; D009599:Nitroprusside; D009569:Nitric Oxide; D006427:Hemin; D006162:Guanylate Cyclase; D008274:Magnesium; D012966:Sodium Cyanide