1887

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Volume 88, Issue 1

Purification and Characterization of a Polyphosphate Kinase from Free

Abstract

SUMMARY: Polyphosphate kinase, an enzyme which incorporated the -phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from by ammonium sulphate fractionation, DEAE-cellulose column chromatography and Sephadex G-200 gel filtration. The enzyme had a broad pH optimum at 6·0 to 7·0 and required Mn or Mg, histone, and inorganic phosphate for activity. The for Mn-ATP was 0·53 m, and for inorganic phosphate was 1·67 m.

Free ATP concentrations greater than 8 inhibited the enzyme. Free Mn or Mg concentrations greater than 2 m or 6 m, respectively, were also inhibitory. Activity was strongly inhibited by 4 m-ADP, 1 m-PP or 20 m-NaF. The effect of ADP might have resulted from reversing the equilibrium of the kinase reaction. The activation by phosphate ions might indicate a role for the enzyme in regulating intracellular phosphate levels or maintaining a phosphorus reserve. The level of enzymic activity in the bacteria responded to changes in inorganic phosphate concentration in the medium. Basic proteins, such as protamine, could substitute for histone as activator. Proteins such as casein or bovine serum albumin would also substitute for histone but only in the absence of inorganic phosphate. The presence of a protein might be necessary to form a complex with the product, thus preventing reversal of the reaction

The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at 100 °C. It behaved as a long-chain-polyphosphate molecule on chromatography in an Ebel’s solvent. The enzymic activity was therefore not that of a protein kinase.

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© Society for General Microbiology, 1975
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1975-05-01
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Purification and Characterization of a Polyphosphate Kinase from Arthrobacter atrocyaneus
Microbiology 88, 65 (1975); https://doi.org/10.1099/00221287-88-1-65
/content/journal/micro/10.1099/00221287-88-1-65
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