Doucet J P, Tuana B S
Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
J Biol Chem. 1991 Sep 15;266(26):17613-20.
The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.
研究了骨骼肌亚细胞组分中低分子量GTP结合蛋白的存在情况。将骨骼肌匀浆、横管、三联体、肌浆网膜和胞质溶胶组分进行十二烷基硫酸钠凝胶电泳分离，并印迹到硝酸纤维素膜上。通过将这些印迹膜与[α-32P]GTP孵育来探究GTP结合蛋白的存在。在所有检测的组分中，GTP标记了分子量为23,000和29,000的两种多肽。[α-32P]GTP的结合具有特异性且依赖于Mg2+。尽管23-kDa和29-kDa多肽在横管组分中均有富集，但23-kDa多肽被[α-32P]GTP标记的程度高于29-kDa多肽。一种40 kDa的GTP结合多肽也在横管制剂中富集，并通过抗Giα免疫染色鉴定为Giα。使用印迹覆盖法和[α-32P]GTP标记的胞质组分，鉴定出了几种与23-kDa和29-kDa GTP结合蛋白相互作用的多肽。这些组分包括分子量为60,000、47,000、44,000、42,000和38,000的多肽，它们主要来源于胞质溶胶，但也与三联体和横管膜相关。发现47-kDa、44-kDa、42-kDa和38-kDa多肽分别与糖酵解酶烯醇化酶、3-磷酸甘油酸激酶、醛缩酶和3-磷酸甘油醛脱氢酶在结构上相关。纯化的糖酵解酶在变性和非变性条件下均能特异性结合23-kDa和29-kDa GTP结合蛋白。GTP结合蛋白与这些多肽的结合对去污剂如3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）、Triton X-100和吐温具有抗性。从嗜铬细胞纯化的23-kDa GTP结合蛋白与三联体和嗜铬细胞膜中的一种157-kDa多肽结合。157-kDa多肽是这些膜中的次要成分，与二氢吡啶受体的亚基无关。鉴于低分子量GTP结合蛋白在膜通讯和分泌偶联等过程中的推测功能，本文讨论了这些蛋白与骨骼肌横管和三联体的关联在信号传递中的作用。
