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

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Abstract
The Malachite Green method for determination of inorganic phosphate (P1 (Itaya K. & Ui, M. (1966) Clin. Chim. Acta 14, 361–366) was modified to measure P 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 mm incubation at 25°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 mr MgCl2 pH 8.0 at 25°C for fresh enzyme preparations.