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澄清 无机聚磷酸盐(Poly P)在新陈代谢中的作用。一种方法具有 通过它可以将未降解的原生Poly P从 舍尔曼丙酸杆菌及其多聚P蛋白激酶和多聚P酶 已经分离出葡萄糖激活剂。已经开发了一种用于测量尺寸的程序 聚P的链长可达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， 将对引发、延伸和终止进行研究。这个 伸长已经被证明是一种过程机制，即合成 在没有从酶中释放的情况下发生，直到链长度约为725 达到了磷酸盐。对于引发剂，PI和短链Poly P将 用作底漆。初步结果表明，短Poly P是一种 并将所得的长链末端标记。长度在以下情况下会有所不同 底物的浓度是不同的。提出了一种使用 [32P]用于确定合成和形成的Poly P长度的Poly P底物 关于终止合同的研究。(2)聚P-葡萄糖激酶：该酶催化 Poly P和ATP对葡萄糖的磷酸化作用，其活性为4 用Poly P进行的抑制研究将确定是否 ATP和Poly P在酶上占据相同的活性部位。用于研究 过程中，长链Poly P末端标记有32P-引物将是 用作衬底。如果机制是过程性的，50%的葡萄糖 应该被标记的末端磷酸化。这一预测将是 测试过。(3)聚P葡糖激酶与Poly P的全长结合 多聚P-激酶可能只在一端。将使用电子显微镜 观察Poly P与酶结合的长链情况。(4)一种方法 为了测定Poly P的分子量，将开发基于 Poly P和DNA电泳过程中相对迁移率的研究 不同大小的。(5)对沙门氏菌的研究：沙门氏菌的放射性 Poly P、葡萄糖-6-P和ATP将使用细胞进行测量，在细胞中 Poly P已经被标记了32PI，作为一种确定 Poly P对葡萄糖的磷酸化在生理上发生。 多聚磷酸酶将被分离和鉴定。(6)动物体内的Poly P 细胞：不同时期肝细胞核内Poly P的大小和含量 将确定再生的阶段及其与蛋白质的相互作用 检查过了。如果存在，将分离和鉴定多聚P激酶。 原子核中的Poly P与核速率的可能关系 将对笔录进行检查。