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 i s to investigate the binding of poly (P) is clearly visible by electron microscopy.
The aim i s to investigate the binding of poly (P) is clearly visible by electron microscopy.
The aim i s 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.
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