Purification and characterization of a polyphosphate kinase from Arthrobacter atrocyaneus.
Polyphosphate kinase, an enzyme which incorporated the gamma-phosphate of ATP into long-chain polyphosphate molecules, was purified more than 700-fold from Arthrobacter atrocyaneus by ammonium sulphate fractionation, DEAE-cellulose column chromatography and Ssphadex G-200 gel filtration. The enzyme had a broad pH optimum at 6-0 to 7-0 and required Mn2+ or Mg2+, histone, and inorganic phosphate for activity. The Km for Mn-ATP was 0-53 mM, and for inorganic phosphate was 1-67 mM. Free ATP concentrations greater than 8 muM inhibited the enzyme. Free Mn2+ or Mg2+ concentrations greater than 2 mM or 6 mM, respectively, were also inhibitory. Activity was strongly inhibited by 4 mM-ADP, 1 mM-PP1 or 20 mM-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 albunim 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 in vitro. The reaction product was characterized, and found to be labile in hydroxylamine, base, and acid at 100 degrees 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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000645:Ammonium Sulfate; D001173:Arthrobacter / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006657:Histones / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000096:biosynthesis; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism; D011108:Polymers; D011479:Protamines / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013010:Sonication
D002364:Caseins; D006657:Histones; D009943:Organophosphorus Compounds; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011108:Polymers; D011479:Protamines; D012710:Serum Albumin, Bovine; D008345:Manganese; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D008274:Magnesium; D000645:Ammonium Sulfate