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

J Biochem. 1986 May;99(5):1465-72. doi: 10.1093/oxfordjournals.jbchem.a135616.

Abstract

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.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism*
  • Animals
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Myosin Subfragments
  • Myosins / metabolism*
  • Osmolar Concentration
  • Peptide Fragments / metabolism*
  • Phosphates / analysis*
  • Rabbits
  • Rosaniline Dyes*
  • Spectrophotometry
  • Temperature

Substances

  • Myosin Subfragments
  • Peptide Fragments
  • Phosphates
  • Rosaniline Dyes
  • malachite green
  • Adenosine Triphosphate
  • Adenosine Triphosphatases
  • Myosins