Membrane-associated nucleoside diphosphate kinase: purification and its role in hormone sensitive adenylate cyclase system

本实验室以前的研究表明，膜相关核苷二磷酸激酶(m-NDP Kinase)可能通过以激素依赖的方式将腺苷环化酶调节的关键辅助因子GTP引导到GTP结合蛋白(Gs)中，从而在腺苷环化酶的调节中发挥作用。为了了解m-NDP激酶的真正作用，在本研究中，从大鼠肝脏纯化的质膜中溶解和纯化m-NDP激酶，并与从相同组织中纯化的胞浆酶(S-NDP激酶)进行比较。测定的部分物理性质为：相对分子质量(单体)为18,300；沉降系数(S)为6.2s；等电点(PI)为6.0。这些数值和动力学参数与S-NDP125-gt；激酶的这些数值和动力学参数几乎相同，后者的特性得到了更全面的研究。I-标记的m-和S-NDPK的肽谱研究给出了基本相同的模式…更多的S。制备了针对S-NDPK的多克隆抗体，该多克隆抗体也与m-NDPK发生交叉反应。用亲和纯化的抗体进行免疫印迹研究表明，纯化的m-和S-NDPK的单体分子量与膜和粗提物中未纯化的酶的值相同。这些结果表明，纯化的m-NDPK在溶解和纯化过程中没有发生明显的修饰，而m-和S-NDPK在蛋白质结构上非常相似，尽管有差异。接下来，通过辛基葡萄糖苷提取，免疫沉淀和亲和纯化的单特异性抗-NDP激酶抗体，检测m-NDPK是否与大鼠肝细胞膜上的胰升糖素和肾上腺素能激动剂敏感的腺苷环化酶系统的组分(GTP结合蛋白(Gs))直接相互作用。结果表明，m-NDP激酶和Gs是以络合物形式提取的，并且这种络合物的形成可通过细胞表面受体被激素可逆地调节，而激素有能力引起大鼠肝脏腺苷环化酶的激活。这些结果表明，m-NDP激酶与Gs之间的相互作用可能是GTP在膜信号转导中的一种作用机制。较少

Previous studies from this laboratory have proposed that membrane-associated nucleoside diphosphate kinase(m-NDP kinase) may play a role in regulation of adenylate cyclase by channeling GTP, an essential cofactor of adenylate cyclase regulation, into GTP binding protein(Gs) in a hormone dependent manner. To understand the true role of m-NDP kinase, in the present study, the m-NDP kinase was solubilized and purified to apparent homogenerity from rat liver purified plasma membranes, and characterized in comparison with the cytosolic enzyme purified from the same tissue(s-NDP kinase). Some physical properties determined were; molecular weight(monomer), 18,300; sedimentation coefficient(s_<20,w>), 6.2s; isoelectric point(pI), 6.0. These values and kinetic parameters of the m-NDP kinase were almost identical to those of the s-NDP_<125>kinase whose characteristics were more externsively studied. A peptide mapping study of the I-labelled m- and s-NDP kinases gave essentially identical pattern … More s. Polycolonal antibodies against the s-NDP kinase, which also cross-reacted with the m-NDP kinase, were prepared. Immunoblotting studies with the affinity-purified antibodies revealed that the monomer molecular weight of the purified m- and s-NDP kinases was identical to the values of unpurified enzymes present in membranes and crude extract. These results demonstrate that the purified m-NDP kinase underwent no remarkable modification during solubilization and purification, and that the m- and s-NDP kinases are quite similar in protein structure, if at all different.Next, whether the m-NDP kinase has a direct interaction with the component(GTP-binding protein(Gs)) of the glucagon- and <beta>-adrenergic agonist-sensitive adenylate cyclase systems in rat liver membranes was examined by extraction with octylglucoside, followed by immunoprecipitation by affinity-purified monospecific anti-NDP kinase antibodies. The results demonstrated that the m-NDP kinase and the Gs were extractable as a complexed form and that the complex formation was reversibly regulated, through cell surface receptors, by hormones which had an ability to cause activation of the rat liver adenylate cyclase. Also, it was suggested that guanine nucleotides rather than hormones were primary regulators of the m-NDP kinase-Gs interaction.These results suggest that the interaction between m-NDP kinase and Gs may play a role as a GTP channeling mechanism in membrane signal transduction. Less