Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside.
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.
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
D005292:Ferricyanides; D009599:Nitroprusside; D009569:Nitric Oxide; D006427:Hemin; D006162:Guanylate Cyclase; D008274:Magnesium; D012966:Sodium Cyanide