Observations on the kinetics, subunit composition, and sulfhydryl reactivity of myosin from Physarum polycephalum

Abstract

A highly purified preparation of myosin from Physarum polycephalum has been shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis to contain heavy chains and only one molecular weight class of light chains, of approx. 15 000 daltons. Kinetic investigations of the Ca 2+-ATPase and Mg 2+ATPase (ATP phosphohydrolases, EC 3.6.1.3) at pH 8.0 gave K m and V values of 17.3 μM and 1.25 μmol P i/min per mg, and 2.4 μM and 0.12 μmol P i/min per mg, respectively. Adenylyl imidodiphosphate, a β-γ-imido ATP analog, inhibited the ATPase activity of Physarum myosin competitively with K i values equal to 350 and 12 μM in the presence of Ca 2+ and Mg 2+, respectively. The ATPase activity of Physarum myosin was inhibited at a very low rate ( t 1 2 = 24 h ) by the ATP analog, 6,6′-dithiobis(inosinyl imidodiphosphate), with concentrations of inhibitor previously shown to inactivate ( t 1 2 ≈ 10 min ) skeletal and cardiac myosins rapidly by reacting with key cysteines.