ROLE OF INORGANIC POLYPHOSPHATES IN METABOLISM

无机多磷酸盐在新陈代谢中的作用

• 批准号: 3277243
• 负责人: HARLAND G. WOOD
• 金额: $ 20.65万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277243
• 关键词: Bacillus stearothermophilus; Propionibacteriaceae; biochemical evolution; chemical chain length; electron microscopy; enzyme mechanism; enzyme structure; gel electrophoresis; glucokinase; goats; laboratory rabbit; laboratory rat; liver cells; liver metabolism; liver regeneration; microorganism metabolism; phosphorylase kinase; polyphosphates; protein sequence; radiotracer; spectrometry; tissue /cell culture; yeasts

The immediate objective is to characterize the enzymes of polyphosphates (poly (P)) metabolism and then apply this information to the ultimate objective of elucidating the role of poly (P) in vivo. The studies will consist of 6 parts. (1) Polyphosphate kinase. A procedure has been developed by which poly (P) is clearly visible by electron microscopy. The aim is to investigate the binding of poly (P) is clearly visible by electron microscopy. The aim is to investigate the binding of poly (P) is clearly visible by electron microscopy. The aim is to investigate the binding of poly (P) to the enzyme and visualize the poly (P) and enzyme during the progressive lengthening of the chain. We also will investigate the amino acid sequence of the ATP binding site of poly (P) kinase. (2) Polyphosphate glucokinase. Propionibacterium shermanii and Nocardia minima are classified in the order Acetinomycetales which are considered to be primitive forms of life. Poly (P) glucokinase utilizes either poly (P) or ATP and N. minima contains another enzyme utilizing only ATP. N. minima may represent an example in a single organism of a transition from poly (P) to ATP as a phosphorylating agent. Glucokinase of Bacillus stearothermophilus, yeast hexokinase and of rat liver will also be investigated. The amino acid sequences will be determined using 5-p-fluorosulfonylbenzoyl adenosine as the labeling agent. The purpose will be to determine if there are similarities in the ATP sites which indicate an evolutionary continuity from ancient to more recent evolutionary forms. In addition, an attempt will be made to label and sequence the poly (P) site of poly (P) glucokinase. (3) Endopolyphosphatases. This enzyme or enzymes will be purified from yeast and it will be determined whether or not there is specificity for certain sizes of poly (P) and whether the cleavages yield certain lengths of poly (P). (4) Exopolyphosphatases. It has been shown that some factor is present in short-chain poly (P) which is required for the utilization of long-chain poly (P). This factor will be identified. (5) Role of poly (P) in vivo in P. shermanii. Using 32 P i , the poly (P) will be prelabeled by growing the bacteria in a lactate medium. The cells will be transferred a glucose medium containing unlabeled P i. The specific radioactivity of the glucose-6 poly (P) and the lambda-P of ATP will be determined to ascertain whether poly (P) serves as the phosphorylating agent in vivo. (6) Poly (P) in animal cells. Investigations will be continued to isolate and conclusively demonstrate that poly (P) is present in animals.

近期的目标是确定多聚磷酸盐的酶的特性。 (Poly(P))新陈代谢，然后将这一信息应用于终极 目的阐明Poly(P)在体内的作用。这些研究将 由6个部分组成。(1)多聚磷酸酶。已经完成了一项程序 通过电子显微镜可以清楚地看到聚(P)的发展。 目的是研究聚(P)的结合是清楚可见的 电子显微镜。目的是研究聚(P)的结合作用 在电子显微镜下清晰可见。其目的是调查 Poly(P)与酶的结合，并可视化Poly(P)和 在链的逐渐加长过程中的酶。我们也会 聚(P)-三磷酸腺苷结合部位的氨基酸序列研究 激活剂。(2)多聚磷酸葡萄糖激酶。舍尔曼丙酸杆菌和 微小诺卡菌被归入醋酸菌目，它们是 被认为是原始的生命形式。聚(P)葡糖激酶利用 Poly(P)或ATP和N.Mina都含有另一种酶，利用 只有三磷酸腺苷。N.Minima可以代表一个例子，在一个生物体中 从聚(P)到作为磷酸化试剂的ATP的过渡。葡萄糖激活剂 嗜热脂肪芽孢杆菌、酵母菌己糖激酶和大鼠肝脏Will 也被调查。氨基酸序列将用以下方法确定 以5-对氟磺酰基苯甲酰腺苷为标记剂。目的 将确定在哪些ATP站点中是否有相似之处 表示从古代到近代的进化连续性 进化的形式。此外，还将尝试将标签和 测定聚(P)葡萄糖激酶的聚(P)位序列。(3) 内切多聚磷酸酶。这一种或几种酶将从 酵母菌，将确定是否有特异性 聚(P)的某些大小以及裂解是否产生特定的 多边形(P)的长度。(4)胞外多聚磷酸酶。事实证明， 一些因素存在于短链聚(P)中，这是 长链聚(P)的利用。这一因素将被确定。 (5)Poly(P)在体内的作用。使用32P I，聚 (P)将通过在乳酸介质中培养细菌来预先标记。这个 细胞将被转移到含有未标记PI的葡萄糖培养液中。 葡萄糖-6聚(P)和λ-P的比放射性 ATP将被确定为Poly(P)是否作为 体内的磷酸化试剂。(6)动物细胞内的Poly(P)。 调查将继续进行，以隔离并最终证明 这种多聚(P)存在于动物体内。