The initial phosphate burst in ATP hydrolysis by myosin and subfragment-1 as studied by a modified malachite green method for determination of inorganic phosphate.
The Malachite Green method for determination of inorganic phosphate (Pi) (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361-366) was modified to measure Pi in the range of 0.2-15 nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6 M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2 g of sodium molybdate, 0.3 g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in 1 liter of 0.7 M HCl, and the absorbance at 650 nm is then measured after a 35-40 min incubation at 25 degrees C. Owing to the high sensitivity and simplicity of the modified method, the slow time course of myosin ATP hydrolysis in the presence of Mg2+ and the size of initial phosphate burst can be determined accurately using relatively low concentrations of native myosin and its subfragment-1. The phosphate burst size varied with changes in pH, ionic strength, and temperature. A typical value was 0.8-0.9 mol per site in 0.1 M KCl, 10 mM MgCl2, pH 8.0 at 25 degrees C for fresh enzyme preparations.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis*; D011817:Rabbits; D012394:Rosaniline Dyes; D013053:Spectrophotometry; D013696:Temperature
D015879:Myosin Subfragments; D010446:Peptide Fragments; D010710:Phosphates; D012394:Rosaniline Dyes; C005095:malachite green; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins