Reversible inactivation of guanylate cyclase by mixed disulfide formation.
Reversible inactivation of guanylate cyclase by mixed disulfide formation.
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DOI：
10.1016/s0021-9258(19)69708-5
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发表时间：
1981-03
期刊：
影响因子：
--
通讯作者：
Brandwein Hj;J. Lewicki;F. Murad
中科院分区：
文献类型：
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作者：
Brandwein Hj;J. Lewicki;F. Murad
从大鼠肺的鸟苷酸环化酶的高度纯化的制剂被灭活的几个二硫化合物在时间和剂量依赖性的方式。胱胺和胱氨酸是测试的最有效的二硫化物，但是含有半胱胺部分（NH 2CH 2CH 2S-）的其他化合物，包括泛硫乙胺和氧化辅酶A，也能够部分地抑制酶。除了基础活性的降低（用Mg 2 +-GTP或Mn 2 +-GTP测量）外，二硫化物抑制的酶在较小程度上被一氧化氮激活。二硫苏糖醇或其他还原剂的治疗恢复了基础活性，并增加了一氧化氮激活后cGMP的产生水平。对照酶样品在Mn 2+或Mg 2+的情况下分别表现出25 μ M或150 μ M的单个GTP Km。然而，使用任一金属离子作为辅因子，胱胺处理的酶显示出这些相同的Km值以及2至3 μ M的额外GTP Km。当[35 S]胱氨酸与纯化的酶一起孵育时，放射性被掺入三氯乙酸可沉淀的蛋白质中，并且在二硫苏糖醇处理后释放计数。此外，[35 S]胱氨酸标记的酶与天然鸟苷酸环化酶在非变性聚丙烯酰胺凝胶上共迁移。这些数据表明，鸟苷酸环化酶和某些天然化合物之间可以形成混合二硫键，并且二硫键的形成导致酶活性的可逆丧失。
Highly purified preparations of guanylate cyclase from rat lung were inactivated by several disulfide compounds in a time- and dose-dependent manner. Cystamine and cystine were the most potent disulfides tested, but other compounds which contained the cysteamine moiety (NH2CH2CH2S-), including pantethine and oxidized coenzyme A, were also able to partially inactivate the enzyme. In addition to the decrease in basal activity (measured with either Mg2+-GTP or Mn2+-GTP), disulfide-inhibited enzyme was activated to a lesser extent by nitric oxide. Treatment with dithiothreitol or other reducing agents restored basal activity and increased the level of cGMP production following nitric oxide activation. Control enzyme samples exhibited a single GTP Km of 25 microM or 150 microM with Mn2+ or Mg2+, respectively. However, cystamine-treated enzyme showed these same Km values as well as an additional GTP Km of 2 to 3 microM using either metal ion as cofactor. When [35S]cystine was incubated with purified enzyme, radioactivity was incorporated into the trichloroacetic acid-precipitable protein, and the counts were released following dithiothreitol treatment. In addition, [35S]cystine-labeled enzyme co-migrated with native guanylate cyclase on nondenaturing polyacrylamide gels. These data indicate that mixed disulfides can be formed between guanylate cyclase and certain naturally occurring compounds, and that disulfide formation leads to a reversible loss of enzyme activity.