Countaway J L, Waddell I D, Burchell A, Arion W J
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1988 Feb 25;263(6):2673-8.
The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
微粒体葡萄糖-6-磷酸酶系统的磷酸水解酶成分已通过对稳态水解过程中形成的磷酰化酶中间体进行32P标记，被鉴定为一种36.5 kDa的多肽。当将破碎的大鼠肝微粒体与该酶的三种不同32P标记底物（葡萄糖-6-磷酸、甘露糖-6-磷酸和焦磷酸）一起孵育，并用三氯乙酸终止反应时，一种36.5 kDa的多肽被标记。用[32P]葡萄糖-6-磷酸标记磷酰化酶中间体被几种已充分表征的葡萄糖-6-磷酸酶活性竞争性抑制剂（如Al(F)4和磷酸根离子）以及热失活所阻断，在用32Pi和[U-14C]葡萄糖-6-磷酸孵育后未观察到标记。与稳态规律一致，[32P]磷酰中间体的量与在完整和破碎的肝微粒体中各种条件下测得的稳态葡萄糖-6-磷酸酶活性直接且定量成正比。标记的36.5 kDa多肽被用针对部分纯化的大鼠肝酶在绵羊中产生的抗血清特异性免疫染色，并且该抗血清从胆酸盐增溶的大鼠肝微粒体中定量免疫沉淀葡萄糖-6-磷酸酶活性。[32P]葡萄糖-6-磷酸也在来自绵羊、兔子、豚鼠和小鼠的肝微粒体以及大鼠肾微粒体中标记了一个大小相似的多肽。葡萄糖-6-磷酸酶似乎是肝内质网的一种次要蛋白质，约占微粒体膜蛋白总量的0.1%。发现十二烷基硫酸钠增溶的膜蛋白的离心是通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离放射性标记的微粒体蛋白的关键步骤。
