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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029569-07
Application #
3277241
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-07-01
Project End
1989-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Phillips, N F; Hsieh, P C; Kowalczyk, T H (1999) Polyphosphate glucokinase. Prog Mol Subcell Biol 23:101-25
Phillips, N F; Li, Z; Lindmark, D G (1997) Isolation of a pyrophosphate-dependent phosphofructokinase from Hexamita inflata. Mol Biochem Parasitol 90:377-80
Hsieh, P C; Shenoy, B C; Samols, D et al. (1996) Cloning, expression, and characterization of polyphosphate glucokinase from Mycobacterium tuberculosis. J Biol Chem 271:4909-15
Hsieh, P C; Kowalczyk, T H; Phillips, N F (1996) Kinetic mechanisms of polyphosphate glucokinase from Mycobacterium tuberculosis. Biochemistry 35:9772-81
Kowalczyk, T H; Horn, P J; Pan, W H et al. (1996) Initial rate and equilibrium isotope exchange studies on the ATP-dependent activity of polyphosphate Glucokinase from Propionibacterium shermanii. Biochemistry 35:6777-85
Hsieh, P C; Shenoy, B C; Jentoft, J E et al. (1993) Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme. Protein Expr Purif 4:76-84
Phillips, N F; Horn, P J; Wood, H G (1993) The polyphosphate- and ATP-dependent glucokinase from Propionibacterium shermanii: both activities are catalyzed by the same protein. Arch Biochem Biophys 300:309-19
Hsieh, P C; Shenoy, B C; Haase, F C et al. (1993) Involvement of tryptophan(s) at the active site of polyphosphate/ATP glucokinase from Mycobacterium tuberculosis. Biochemistry 32:6243-9
Kowalczyk, T H; Phillips, N F (1993) Determination of endopolyphosphatase using polyphosphate glucokinase. Anal Biochem 212:194-205
Phillips, N F (1988) The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate. Biochemistry 27:3314-20

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