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
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
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DOI：
10.1016/0304-4165(82)90008-3
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
WOLIN, MS
中科院分区：
文献类型：
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作者：
IGNARRO, LJ;DEGNAN, JN;WOLIN, MS
牛肺可溶性鸟苷酸环化酶被纯化到明显的同质形式，缺乏血红素。血红素缺乏型鸟苷酸环化酶与血红素反应，在过量的二硫苏糖醇的存在下，然后通过凝胶过滤去除未结合的血红素，迅速和容易地重建与血红素。结合血红素光谱验证和NO转移的吸光度最大值的方式特征的其他血红素。血红素缺乏和血红素重建鸟苷酸环化酶进行了比较，酶完全保留血红素在纯化过程中。NO和S-亚硝基-N-乙酰青霉胺仅轻微激活血红素缺乏的鸟苷酸环化酶，但显着激活血红素重建和含血红素形式的酶。通过在含有二硫苏糖醇的酶反应混合物中加入1 μ M血红素，实现血红素缺陷型鸟苷酸环化酶的显著活化的恢复。预先形成的NO-血红素激活所有形式的鸟苷酸环化酶在没有额外的血红素。在MgGTP或MnGTP存在下观察到鸟苷酸环化酶活化，尽管酶活化的幅度始终大于MgGTP。对于未活化的鸟苷酸环化酶，在过量Mn 2+或Mg 2+存在下GTP的表观Km分别为10 μ M和85-120 μ M。在Mn 2+存在下GTP的表观Km没有改变，但是在Mg 2+存在下的Km用活化的酶降低到58 μ M。在Mg ~（2+）或Mn ~（2+）存在下，酶激活剂可增加Vmax。在这项研究中报道的数据表明，纯化的鸟苷酸环化酶结合血红素和后者是需要酶激活NO和亚硝基化合物。
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 .mu.M 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 .mu.M and 85-120 .mu.M, 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 .mu.M with activated enzyme. Vmax was 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.