A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia.
Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported.
Mullan
Alan
A
School of Biology and Biochemistry and QUESTOR Centre, The Queen's University of Belfast, Medical Biology Centre, Ireland.
Quinn
John P
JP
McGrath
John W
JW
D016956:Burkholderia cepacia / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D011122:Polyphosphates / Q000378:metabolism*; D014048:Tolonium Chloride / Q000737:chemistry / Q000378:metabolism
D011122:Polyphosphates; D014048:Tolonium Chloride; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase