Soluble guanylate cyclase from rat lung exists as a heterodimer.
Soluble guanylate cyclase from rat lung exists as a heterodimer.
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DOI：
10.1016/s0021-9258(17)38380-1
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发表时间：
1986-06
期刊：
影响因子：
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通讯作者：
Y. Kamisaki;S. Saheki;M. Nakane;J. Palmieri;T. Kuno;B. Chang;S. Waldman;F. Murad
中科院分区：
文献类型：
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作者：
Y. Kamisaki;S. Saheki;M. Nakane;J. Palmieri;T. Kuno;B. Chang;S. Waldman;F. Murad
用一步免疫亲和层析法将大鼠肺可溶性鸟苷酸环化酶（EC 4.6.1.2）纯化至均一。纯化的可溶性鸟苷酸环化酶的比活性为432和49.1 nmol的环GMP形成每分钟/毫克蛋白质与锰和镁离子作为辅因子，分别。这表示纯化约2，000倍，回收率为50%。天然酶具有150，000的分子量和4.8nm的斯托克斯半径，如在Spherogel TSK-G3000 SW凝胶渗透色谱法上测定的。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳产生分子量为82，000和70，000的两条蛋白质染色带。纯化的可溶性鸟苷酸环化酶也进行了非变性聚丙烯酰胺凝胶电泳，等电聚焦电泳，离子交换层析，和GTP-琼脂糖亲和层析。这些额外的纯化程序证实了与酶活性一致的单个蛋白质峰的存在。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上分离的两个亚基被证明具有不同的一级结构，通过免疫印迹与纯化的可溶性鸟苷酸环化酶制备的单克隆和多克隆抗体，并通过肽图谱与木瓜蛋白酶或金黄色葡萄球菌V8蛋白酶处理。这些数据表明，从大鼠肺中纯化的可溶性鸟苷酸环化酶是由具有不同一级结构的82，000-和70，000-道尔顿亚基组成的异源二聚体。
The soluble form of guanylate cyclase (EC 4.6.1.2) from rat lung has been purified to homogeneity by a one-step immunoaffinity chromatographic procedure. The purified soluble guanylate cyclase has specific activities of 432 and 49.1 nmol of cyclic GMP formed per min/mg protein with manganese and magnesium ions as a cofactor, respectively. This represents a purification of approximately 2,000-fold with a 50% recovery. The native enzyme has a molecular weight of 150,000 and a Stokes radius of 4.8 nm as determined on Spherogel TSK-G3000SW gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with molecular weights of 82,000 and 70,000. The purified soluble guanylate cyclase was also subjected to native polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, ion exchange chromatography, and GTP-agarose affinity chromatography. These additional purification procedures confirmed the presence of a single protein peak coincident with enzyme activity. The two subunits separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were shown to have different primary structures by immunoblotting with monoclonal and polyclonal antibodies prepared against purified soluble guanylate cyclase and by peptide mapping with papain or Staphylococcus aureus V8 protease treatment. These data demonstrate that soluble guanylate cyclase purified from rat lung is a heterodimer composed of 82,000- and 70,000-dalton subunits with different primary structures.