Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung.
Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D009569:Nitric Oxide / Q000494:pharmacology*; D011485:Protein Binding; D012995:Solubility
D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase