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.

这些调查是其最终目标的阐明 无机多磷酸盐（聚P）在代谢中的作用。 一种方法 设计了可以从中可以从中隔离的原生聚P 谢尔马尼丙肽和酶，聚P激酶和聚P 葡萄糖酶已经分离出来。 已经开发了尺寸的程序 poly P到链长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， 将研究启动，伸长和终止。 这 伸长率已被证明是一种过程机制，即合成 发生不从酶释放到约725的链长度 达到磷酸盐。 为了开始，PI和短链聚P是 用作底漆。 初步结果表明短多p用作 底漆和由此产生的长链被标记。 长度会有所不同 引物的浓度变化。 使用一种方法提出了一种方法 [32p] poly P底漆确定合成poly P的长度，并 终止研究。 （2）聚P葡萄糖激酶：这种酶催化 葡萄糖与Poly P和ATP的磷酸化，活性为4 通过聚P进行抑制作用的时间更大，以确定是否是否 ATP和Poly P占据酶上的同一活性位点。 用于研究 处理过程，用32p-primer标记的长链poly P End将是 用作基材。 如果该机制是过程，则葡萄糖的50％ 应由标记的端磷酸化。 这个预测将是 测试。 （3）聚P葡萄糖酶与全长Poly P和 聚P激酶可能仅在一端。 电子显微镜将被使用 观察链poly P与酶结合多长时间。 （4）一种方法 为了确定Poly P的分子量，将基于 关于聚P和DNA电泳期间迁移的相对速率 大小不同。 （5）对Shermanii的研究： Poly P，葡萄糖-6-P和ATP将使用细胞进行测量 Poly P已标记为32PI作为确定是否是否 通过生理发生葡萄糖的磷酸化。 多磷酸酶将被分离并表征。 （6）动物中的聚P 细胞：各种肝核中聚P的大小和含量 将确定再生阶段，并与蛋白质相互作用 检查。 如果存在，聚P激酶将被隔离并表征。 核中聚P与核速率之间的可能关系 将检查转录。