A PORCINE BRAIN PROTEIN (35K-PROTEIN) WHICH BUNDLES MICROTUBULES AND ITS IDENTIFICATION AS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
A PORCINE BRAIN PROTEIN (35K-PROTEIN) WHICH BUNDLES MICROTUBULES AND ITS IDENTIFICATION AS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
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DOI：
10.1093/oxfordjournals.jbchem.a134260
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发表时间：
1983-01-01
影响因子：
2.7
通讯作者：
SAKAI, H
中科院分区：
文献类型：
--
作者：
KUMAGAI, H;SAKAI, H
从猪脑中分离出一种既与微管蛋白结合又与微管蛋白聚合物结合的蛋白质。该蛋白质在SDS[十二烷基硫酸钠]-聚丙烯酰胺凝胶电泳上的分子量为35，000（命名为35 K [千道尔顿]蛋白质）。通过硫酸铵分级沉淀、Sephadex G-100凝胶过滤柱层析、微管蛋白-琼脂糖凝胶亲和柱层析和磷酸纤维素柱层析等步骤纯化了35 K蛋白。35 K蛋白引起显着增强的浊度增加产生的微管蛋白聚合在DMSO [二甲亚砜]的存在下，但没有能力启动聚合的纯微管蛋白在DMSO的情况下。35 K蛋白质以浓度依赖性方式与微管蛋白聚合物共沉积。电镜观察显示微管蛋白聚合物形成束。由于35 K蛋白的作用与微管蛋白聚合偶联，因此在微管蛋白聚合被Ca 2+或秋水仙素抑制的条件下，35 K蛋白不会引起浊度增加。吸附在微管蛋白-Sepharose 4 B上的35 K蛋白通过加入2 mM ATP洗脱。ATP抑制35 K蛋白与微管蛋白二聚体或多聚体的相互作用。根据SDS-聚丙烯酰胺凝胶电泳的迁移率、限制性蛋白水解的裂解模式、与微管蛋白结合的能力等特性，最终将35 K蛋白鉴定为甘油醛3-磷酸脱氢酶。
A protein which binds to both tubulin and tubulin polymer was isolated from porcine brains. This protein has MW of 35,000 on SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis (designated as 35 K [kilodalton] protein). The 35 K protein was purified through several steps of purification including ammonium sulfate fractionation, Sephadex G-100 gel filtration column chromatography, microtubule protein-agarose gel affinity column chromatography and phosphocellulose column chromatography. The 35 K protein caused pronounced enhancement of the turbidity increase produced by tubulin polymerization in the presence of DMSO [dimethylsulfoxide], but did not have the ability to initiate polymerization of pure tubulin in the absence of DMSO. The 35 K protein co-sediments with tubulin polymer in a concentration-dependent manner. EM observation revealed the formation of bundles of tubulin polymer. Since the effect of 35 K protein was coupled with tubulin polymerization, 35 K protein did not cause the turbidity increase under conditions where tubulin polymerization was inhibited by Ca2+ or colchicine. The 35 K protein adsorbed on tubulin-Sepharose 4B was eluted by the addition of 2 mM ATP. ATP inhibited the interaction of 35 K protein with tubulin dimer or polymer. The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.