LIVER PYROPHOSPHATE/PHOSPHOTRANSFERASE ACTIVITIES

肝脏焦磷酸/磷酸转移酶活性

• 批准号: 2134644
• 负责人: ROBERT C NORDLIE
• 金额: $ 13.46万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2134644
• 关键词: SDS polyacrylamide gel electrophoresis; electrofocusing; endoplasmic reticulum; enzyme activity; enzyme complex; enzyme mechanism; glucose 6 phosphatase; immunochemistry; intracellular transport; ion transport; isozymes; laboratory mouse; liver metabolism; malignant ascites; membrane transport proteins; microsomes; phosphates; phosphorylation; pyrophosphates; western blottings

The long-range goal of these studies is to better-understand the structural inter-relationships, metabolic roles, and bioregulation of the bifunctional, multicomponent, membrane bound hepatic glucose-6- phosphatase system (EC 3.1.3.9) under normal, fasted, diabetic, cancerous, and other pathologic conditions. Glucose-6-phosphatase is an organized system within the endoplasmic reticulum consisting of at least six components: The catalytic unit itself, a stabilizing protein, and four (or more) translocases responsible for transport of individual substrates and products to/from the sequestered catalytic unit. Attention will be focused on translocase "T2beta", the transporter for product Pi and substrates PPi and carbamyl-P, more specifically on its response and underlying mechanism in Ehrlich ascites tumor-bearing mice. Answers will be sought to the question of why Pi appears not to be transported from the medium (cytosol) to the lumen of the endoplasmic reticulum even though total T2beta protein increases three-fold in ascites tumor-bearing mice. As a working hypothesis, it is proposed that two T2beta forms exist, i.e , "T2beta" in normal control and "T2beta'" in liver microsomes derived from tumor-bearing mice. Studies are proposed to 1) correlate the time-frame for change in T2beta with tumor development; 2) examine the inhibition kinetics with liver microsomes from humor-bearing and control mice to delineate sites of common and distinct interactions among glucose-6-P, PPi, Pi, carbamyl-P, and glucose, and tumor-related changes therein; 3) determine the specificity and rates of transport of carbamyl-P, PPi, and Pi into, and out of, microsomes derived from tumor-bearing and control mouse liver; and 4) check for multiple forms of translocase T2beta using Western immunoblotting, combined with PAGE without and with isoelectric focusing, and isolate and chemically characterize these separate forms. Experimentation will include a combination of immunochemistry, protein chemistry, enzymology (primarily kinetics), transport studies with a novel fast-sampling, rapid-filtration apparatus, and isotope techniques for study of protein phosphorylation/ dephosphorylation. These studies will give valuable new knowledge of how the gluconeo/genically-stressed tumor-bearing mouse adapts in an attempt to maintain normoglycemia and to conserve glucose-6-P for the liver's use. The studies may also define new foci for anticancer drugs. A better understanding of how components of the glucose-6-phosphatase system function individually and in concert, normally and under such stressed conditions as diabetes, glucocorticoid administration, and the presence of developing tumors, will be achieved.

这些研究的远程目标是更好地理解 结构之间的相互关系，代谢作用和生物调节 双功能，多组分，膜结合的肝葡萄糖-6- 在正常，禁食，糖尿病，磷酸酶系统（EC 3.1.3.9）， 癌和其他病理状况。 葡萄糖-6-磷酸酶是一个 内质网中有组织的系统，至少由 六个成分：催化单元本身，稳定蛋白质和 负责个人运输的四个（或更多）易位 从隔离的催化单元/往/来自底物和产物。 注意将集中于易位酶“ T2Beta”， PPI和Carbamyl-P产品PI和底物，更具体地说 Ehrlich腹水肿瘤小鼠的反应和潜在机制。 将寻求答案的问题，即为什么PI似乎不是 从培养基（细胞质）运输到内质的腔内 即使总T2BETA蛋白增加了三倍，网状 腹水肿瘤小鼠。 作为一个工作假设，有人提出 存在两种T2Beta形式，即正常对照中的“ T2Beta”和“ T2Beta'” 在源自肿瘤小鼠的肝微粒体中。 研究是 提议1）将T2Beta变化的时间框架与肿瘤相关联 发展; 2）检查肝微粒体的抑制动力学 从幽默和对照小鼠到描绘共同点和 葡萄糖-6-P，PPI，PI，Carbamyl-P和 葡萄糖和肿瘤相关的变化； 3）确定特异性 卡氨基-P，PPI和PI进入并从中出发的运输速率 源自肿瘤和对照小鼠肝脏的微粒体；和4） 使用Western检查多种形式的易位酶T2Beta 免疫印迹，与没有以及等电聚焦的页面相结合， 分离和化学表征这些单独的形式。 实验将包括免疫化学，蛋白质的组合 化学，酶学（主要是动力学），运输研究 新颖的快速采样，快速滤发器和同位素技术 用于研究蛋白质磷酸化/去磷酸化。这些研究 将为葡萄糖/植物压力如何提供宝贵的新知识 含肿瘤的小鼠适应试图维持正常血糖和 为肝脏使用葡萄糖-6-P。 研究也可能定义 抗癌药物的新焦点。更好地了解组件的方式 葡萄糖-6-磷酸酶系统的单独和协同功能 通常并在糖尿病等应力条件下，糖皮质激素 将实现给药和发展肿瘤的存在。