Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.
Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide.
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通过孵育或过氧化氢激活大鼠肺中的可溶性鸟苷酸环化酶。
DOI：
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发表时间：
1976
影响因子：
4.8
通讯作者：
P. Lad
中科院分区：
文献类型：
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作者：
A. A. White;K. Crawford;C. Patt;P. Lad
从脂肪肺匀浆制备的37,000 X g上清部分显示，在30度孵育30分钟（预孵育）后，鸟苷酸环化酶活性增加2至3倍。用Triton X-100处理上清部分后，活性增加程度与预孵育大致相同，但预孵育后活性不会增加。通过对Sepharose 2B进行预孵育前后的层析，发现其活性仅与可溶性鸟苷酸环化酶有关，而与颗粒酶无关。预孵育激活需要O2。2-巯基乙醇等硫醇、牛血清白蛋白、KCN和二乙基二硫代氨基甲酸钠均能完全抑制。这些抑制剂表明激活需要铜，并且通过证明20至60 muM CuCl2可以减轻0.1 mM二乙基二硫代氨基甲酸钠的抑制，证实了这一点。2-巯基乙醇的抑制也可以通过去除Sephadex G-25柱上的硫醇来逆转，然而，这种处理部分激活了酶。在预孵育的制剂中加入2-巯基乙醇不会逆转活化。H2O2被发现可以激活鸟苷酸环化酶，或者通过在肺上清中与葡萄糖氧化酶和葡萄糖生成H2O2，或者通过将H2O2添加到过氧化氢酶被KCN抑制的制剂中。KCN或牛血清白蛋白能够部分抑制葡萄糖氧化酶和葡萄糖的激活，然而，大量的葡萄糖氧化酶可以克服这种抑制，这表明Cu2+在低H2O2浓度下具有催化作用。在预孵育过程中没有发现H2O2形成的直接证据，然而，通过分光光度法检测肺上清液中血红蛋白形成的血红蛋白，获得了间接证据。H2O2被认为是由氧合血红蛋白与抗坏血酸反应产生的。
A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.