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

doi:10.1016/0304-4165(82)90008-3

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 718, Issue 1, 17 September 1982, Pages 49-59

https://doi.org/10.1016/0304-4165(82)90008-3 Get rights and content

Abstract

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-containg forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 μM 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 K_m for GTP in the presence of excess Mn²⁺ or Mg²⁺ was 10 μM and 85–120 μM, respectively, for unactivated guanylate cyclase. The apparent K_m for GTP in the presence of Mn²⁺ was not altered but the K_m in the presence of Mg²⁺ was lowered to 58 μM 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 nitroso compounds.

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It is widely accepted that NO binds to the heme group, which transfers the enzyme into the active state. An early and simple strategy to test this was the investigation of heme-free sGC preparations, which showed a lack of NO responsiveness [27]. However, the detailed molecular events associated with NO binding to the heme group and subsequent enzyme activation are complex.
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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

Abstract

J. Biol. Chem.

Blood

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Science

J. Cyclic Nucleopide Res.