Waldman S A, Lewicki J A, Brandwein H J, Murad F
J Cyclic Nucleotide Res. 1982;8(6):359-70.
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.
将大鼠肝脏匀浆105,000×g颗粒组分中的鸟苷酸环化酶（形成20 pmol环鸟苷酸/分钟/毫克蛋白质）在无去污剂的情况下，于37℃用5μg/ml胰蛋白酶孵育12分钟使其溶解。最佳溶解取决于胰蛋白酶和颗粒制剂的浓度。在任何测试的时间点或胰蛋白酶浓度下，几乎未观察到颗粒鸟苷酸环化酶的激活。用胰蛋白酶溶解的鸟苷酸环化酶通过硫酸铵沉淀、GTP亲和层析和制备性聚丙烯酰胺凝胶电泳纯化了约500倍（9.4 nmol/分钟/毫克蛋白质）。活性在Sephadex G - 200上以单峰洗脱（斯托克斯半径 = 40 Å），在蔗糖密度梯度上以单峰迁移（S20,w = 4.6）。因此，胰蛋白酶片段估计分子量约为80,000道尔顿（Mr），摩擦比（f/fo）为1.4。这些部分纯化的制剂与Mn - GTP呈现线性双倒数图，希尔系数为1.0。这与粗制膜相关酶的希尔系数1.5形成对比。膜结合的和胰蛋白酶溶解的鸟苷酸环化酶用一氧化氮激活3倍和4倍，并被1 mM胱胺抑制。7.5 mM二硫苏糖醇可部分逆转胱胺的抑制作用。这些研究表明，通过有限蛋白酶解溶解的颗粒鸟苷酸环化酶适合用“经典”色谱技术进行纯化。部分纯化的片段包含催化位点、一氧化氮激活位点以及活性所需的至少一个巯基。
