ROLE OF INORGANIC POLYPHOSPHATE IN METABOLISM

无机多磷酸盐在代谢中的作用

• 批准号: 3277241
• 负责人: HARLAND G. WOOD
• 金额: $ 13.7万
• 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/3277241
• 关键词: Propionibacteriaceae; cell cycle; electron microscopy; enzyme structure; gel electrophoresis; genetic transcription; glucokinase; 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.

这些调查的最终目标是阐明 无机多磷酸盐（Poly P）在新陈代谢中的作用。 了一种方法 设计了未降解的天然Poly P可以从 谢氏丙酸杆菌和酶，聚P激酶和聚P 葡萄糖激酶已被分离。 已经制定了一个程序， Poly P链长达700。 进一步的研究将包括六个 部分：（1）聚P激酶：聚合物合成的三个阶段， 将研究起始、延伸和终止。 的 已经表明伸长是一种进行性机制，即，合成 直到链长约725 磷酸盐已达到。 对于起始，Pi和短链Poly P将是 用作引物。 初步结果表明，短聚P作为一个 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， [32P]Poly P引物用于测定合成的Poly P的长度， 终止研究。 (2)聚P葡萄糖激酶：这种酶催化 用Poly P和ATP磷酸化葡萄糖，活性为4 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。 为研究 进行性过程中，用32P-引物标记的长链Poly P末端将被 用作衬底。 如果机制是进行性的，50%的葡萄糖 应该被标记的末端磷酸化。 这一预测将是 测试. (3)聚P葡萄糖激酶与聚P的全长结合， 聚P激酶可能仅在一端。 将使用电子显微镜 观察长链Poly P与酶结合的情况。 (4)的方法 用于测定聚P的分子量将根据 对Poly P和DNA电泳过程中迁移的相对速率的影响 不同大小的。 (5)关于P. shermanii的研究： 将使用细胞测量多聚P、葡萄糖-6-P和ATP， Poly P已经用32Pi标记，作为确定是否存在放射性的方法。 葡萄糖被Poly P磷酸化发生在生理学上。 将分离并表征多磷酸酶。 (6)动物中的聚P 细胞：不同时间肝细胞核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 考察 如果存在，将分离并表征聚P激酶。 细胞核内Poly P与细胞核凋亡率的可能关系 转录将被检查。