An in vitro kinetic assay of ATPase by phosphorus-31 NMR.
The ATPase activity of RecA protein was examined by monitoring the changes of NMR phosphorus signals of ATP, ADP and inorganic phosphate. The areas of phosphorus-31 NMR peaks from inorganic phosphate and ADP, which increased with time, and the signals from ATP, which decreased with time, were fitted by a linear least square method to obtain the initial rate constants. The rate constants were examined for dependence on RecA protein concentration, temperature and pH. This assay system is useful for testing extrinsic physico-chemical effects on ATPase activity; because only essential components are present in a confined system, and the rate constants can be measured with a single step.
Chinami
M
M
Department of Virology, Kurume University School of Medicine, Japan.
Shingu
M
M
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011938:Rec A Recombinases / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases