Partial purification and characterization of particulate guanylate cyclase from rat liver after solubilization with trypsin.

S A Waldman, J A Lewicki, H J Brandwein, F Murad
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Abstract

Guanylate cyclase from 105,000 X g particulate fractions of rat liver homogenates (20 pmoles of cyclic GMP formed/min/mg protein) was solubilized in the absence of detergents by incubating fractions 12 min at 37 degrees with 5 ug/ml trypsin. Optimal solubilization was dependent upon trypsin and particulate preparation concentrations. Virtually no activation of particulate guanylate cyclase was observed at any time point or trypsin concentration tested. Guanylate cyclase solubilized with trypsin was purified about 500-fold (9.4 nmoles/min/mg protein) using ammonium sulfate precipitation, GTP-affinity chromatography, and preparative polyacrylamide gel electrophoresis. Activity eluted as a single peak on Sepadex G-200 (Stokes radius = 40 A) and migrated as a single peak on sucrose density gradients (S20,w = 4.6). Thus, the tryptic fragment was estimated to be about 80,000 daltons (Mr) with a frictional ratio (f/fo) of 1.4. These partially purified preparations exhibited linear double reciprocal plots with Mn-GTP and Hill coefficients of 1.0. This is in contrast to the crude membrane-associated enzyme which had a Hill co-efficient of 1.5. Membrane-bound and trypsin-solubilized guanylate cyclase were activated 3- and 4-fold with nitric oxide and were inhibited with 1mM cystamine. Cystamine inhibition could be partially reversed with 7.5 mM dithiothreitol. These studies indicate that particulate guanylate cyclase solubilized by limited proteolysis is amenable to purification by "classical" chromatographic techniques. The partially purified fragment contains the catalytic site, the site for nitric oxide activation, and at least one sulfhydryl group required for activity.

胰蛋白酶溶解大鼠肝脏中颗粒鸟苷酸环化酶的部分纯化和特性研究。
从105,000 X g大鼠肝脏匀浆颗粒中提取的鸟苷酸环化酶(形成的环状GMP为20 pmol /min/mg蛋白)在没有洗涤剂的情况下,与5 ug/ml胰蛋白酶在37度下孵育12分钟。最佳增溶作用取决于胰蛋白酶和颗粒制剂浓度。在任何时间点或胰蛋白酶浓度测试中,几乎没有观察到颗粒鸟苷酸环化酶的激活。经胰蛋白酶溶解的鸟苷酸环化酶,采用硫酸铵沉淀、gtp亲和层析、制备性聚丙烯酰胺凝胶电泳等方法纯化了约500倍(9.4 nmol /min/mg蛋白)。活性在Sepadex G-200 (Stokes半径= 40 a)上呈单峰洗脱,在蔗糖密度梯度(S20,w = 4.6)上呈单峰迁移。因此,估计色氨酸片段约为80,000道尔顿(Mr),摩擦比(f/fo)为1.4。这些部分纯化的制剂呈现线性双倒易图,Mn-GTP和Hill系数为1.0。这与粗膜相关酶的希尔系数为1.5形成对比。膜结合型和胰蛋白酶溶解型鸟苷酸环化酶被一氧化氮激活3倍和4倍,并被1mM半胱胺抑制。7.5 mM二硫苏糖醇可部分逆转半胺抑制作用。这些研究表明,通过有限的蛋白水解溶解的颗粒鸟苷酸环化酶可以通过“经典”色谱技术纯化。部分纯化的片段含有催化位点、一氧化氮活化位点和活性所需的至少一个巯基。
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