ROLE OF INORGANIC POLYPHOSPHATE IN METABOLISM

无机多磷酸盐在代谢中的作用

• 批准号: 3277242
• 负责人: HARLAND G. WOOD
• 金额: $ 14.22万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1989-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277242
• 关键词: Propionibacteriaceae; cell cycle; electron microscopy; enzyme mechanism; enzyme structure; gel electrophoresis; genetic transcription; glucokinase; laboratory rat; liver cells; liver metabolism; liver regeneration; metabolism; microorganism metabolism; phosphorylase kinase; polyphosphates; propionates; radiotracer; spectrometry; tissue /cell culture

These investigations have as their ultimate objective the elucidation of the role of inorganic polyphosphates (Poly P) in metabolism. A method has been devised by which the undegraded native Poly P can be isolated from Propionibacterium shermanii and the enzymes, Poly P kinase and Poly P glucokinase have been isolated. A procedure has been developed for sizing Poly P up to chain lengths 700. Further studies will consist of six parts: (1) Poly P kinase: The three phases of polymer synthesis, initiation, elongation and termination will be investigated. The elongation has been shown to be a processive mechanism, i.e., the synthesis occurs without release from the enzyme until a chain length of about 725 phosphates is reached. For initiation, Pi and short chain Poly P will be used as primers. Preliminary results indicate short Poly P serves as a primer and the resulting long chain is end labeled. The length varies if the concentration of the primer is varied. A method is proposed using a [32P] Poly P primer to determine the length of Poly P synthesized and for study of termination. (2) Poly P glucokinase: This enzyme catalyzes phosphorylation of glucose with both Poly P and ATP, the activity being 4 times greater with Poly P. Inhibition studies will be done to determine if ATP and Poly P occupy the same active site on the enzyme. For study of the processive process, long chain Poly P end labeled with a 32P-primer will be used as a substrate. If the mechanism is processive, 50% of the glucose should be phosphorylated by the labeled end. This prediction will be tested. (3) Poly P glucokinase combines with the full length of Poly P and Poly P kinase probably at one end only. Electron microscopy will be used to observe how long chain Poly P combines with the enzymes. (4) A method for determination of the molecular weight of Poly P will be developed based on the relative rate of migration during electrophoresis of Poly P and DNA of different sizes. (5) Studies with P. shermanii: The radioactivity of the Poly P, glucose-6-P and ATP will be measured using cells in which the Poly P has been labeled with 32Pi as a method of determining if the phosphorylation of glucose by Poly P occurs physiologically. Polyphosphatase will be isolated and characterized. (6) Poly P in animal cells: The size and content of Poly P in hepatic nuclei during various stages of regeneration will be determined and its interaction with protein examined. Poly P kinase will be isolated and characterized if present. The possible relationship between Poly P in nuclei and the rate of nuclear transcription will be examined.

这些调查的最终目的是阐明 无机多磷酸盐（Poly P）在新陈代谢中的作用。 一个方法有 设计了一种方法，可以从其中分离出未降解的天然 Poly P 谢尔曼丙酸杆菌和酶、聚 P 激酶和聚 P 已分离出葡萄糖激酶。 已开发出尺寸确定程序 Poly P 链长可达 700。进一步的研究将包括六项 部分： (1) Poly P 激酶：聚合物合成的三个阶段， 将研究引发、伸长和终止。 这 伸长已被证明是一种进行性机制，即合成 直到链长约为 725 时，酶才会释放 达到磷酸盐。 对于引发，Pi 和短链 Poly P 将是 用作底漆。 初步结果表明短聚 P 作为 引物并对所得长链进行末端标记。 长度变化如果 引物的浓度是变化的。 提出了一种方法，使用 [32P] Poly P 引物，用于确定合成的 Poly P 的长度并用于 终止研究。 (2)聚P葡萄糖激酶：该酶催化 葡萄糖与 Poly P 和 ATP 的磷酸化，活性为 4 Poly P 的效果要大一倍。将进行抑制研究以确定是否 ATP 和 Poly P 占据酶上相同的活性位点。 为了研究 加工过程中，用 32P 引物标记的长链 Poly P 末端将被 用作基材。 如果该机制是进行性的，则 50% 的葡萄糖 应该被标记的末端磷酸化。 这个预测将是 已测试。 (3) Poly P葡萄糖激酶与全长Poly P结合， Poly P 激酶可能仅在一端。 将使用电子显微镜 观察 Poly P 与酶结合的长链。 (4)方法 用于测定 Poly P 分子量的方法将基于 Poly P 和 DNA 电泳过程中相对迁移率的影响 不同尺寸的。 (5) 谢氏 P. shermanii 的研究： Poly P、葡萄糖-6-P 和 ATP 将使用以下细胞进行测量： Poly P 已用 32Pi 标记作为确定是否 Poly P 对葡萄糖的磷酸化在生理上发生。 将分离并表征多磷酸酶。 (6) 动物中的多聚P 细胞：不同时期肝细胞核中 Poly P 的大小和含量 将确定再生阶段及其与蛋白质的相互作用 检查了。 如果存在 Poly P 激酶，将对其进行分离和表征。 核内Poly P与成核率的可能关系 将检查转录。