Winger Jonathan A, Derbyshire Emily R, Marletta Michael A
Department of Medicinal Chemistry, the University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2007 Jan 12;282(2):897-907. doi: 10.1074/jbc.M606327200. Epub 2006 Nov 10.
Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.
可溶性鸟苷酸环化酶（sGC）是主要的一氧化氮（NO）受体，其调节与NO与辅基血红素基团的结合有关。最近的研究表明，sGC激活的程度和持续时间取决于嘌呤核苷酸的存在和比例以及过量NO的存在。我们在37℃和10℃下，在有和没有底物类似物鸟苷-5'-[（α，β-亚甲基）三磷酸]（GMPCPP）或激活剂3-（5'-羟甲基-3'-呋喃基）-1-苄基吲唑（YC-1）的情况下，测量了全长α1β1 sGC以及构建体β1（1-194）、β1（1-385）和β2（1-2）的NO解离情况。每个构建体的NO解离情况都很复杂，需要两个指数函数来拟合数据。降低温度会降低所有构建体中较快指数函数的贡献。在37℃时，加入YC-1适度加速了sGC和β2（）的NO解离，在10℃时显著加速了sGC的NO解离。GMPCPP的存在也在10℃时显著加速了sGC的NO解离。这种加速是由于每个指数函数观察到的速率增加以及较快指数函数的贡献增加。较快指数函数贡献的增加与NO对sGC的更高激活相关。这些数据表明，sGC亚铁-亚硝酰复合物采用两种五配位构象，一种是活性较低的“封闭”形式，其缓慢释放NO，另一种是活性较高的“开放”形式，其快速释放NO。这两种形式的比例影响NO解离的总体速率。这些结果对sGC在体内的功能具有重要意义，在体内有证据表明存在两种受NO调节的活性状态。 （原文中β2(1-217) 括号内容似乎有误，按照正确内容修改后翻译如上，你可根据实际情况调整）
