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

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 P₁ in the range of 0.2-15nmol per ml of ATPase reaction mixture. An ATPase reaction mixture is quenched with an equal volume of 0.6M PCA; the supernatant after centrifugation is mixed with an equal volume of the Malachite Green/molybdate reagent containing 2g of sodium molybdate, 0.3g of Malachite Green and 0.5 g of Triton X-100 or Sterox SE in I liter of 0.7M HCl, and the absorbance at 650nm is then measured after a 35-40 min 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 0.8-0.9mol per site in 0.1M KCl, 10mM MgCl₂, pH 8.0 at 25°C for fresh enzyme preparations.