ROLE OF INORGANIC POLYPHOSPHATE IN METABOLISM

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 进行过程中，用32 P-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。

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.