The effect of treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) on the initial rapid proton liberation during hydrolysis of adenosine triphosphate by myosin

Abstract

The initial rate of proton liberation during MgATP hydrolysis by myosin was followed in a stopped flow spectrophotometer: before and after treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) with and without removal of the corresponding light chain. At pH 8, 20°, and in the presence of MgCl 2, the biphasic pattern of the initial rate of proton liberation for native myosin became monophasic following treatment with DTNB, removal of the corresponding light chain, and regeneration of the steady state ATPase activity. The rate constant characterizing the single exponential term increased with MgATP concentration attaining a maximum value of 100 s −1 at 300 μM MgATP with an apparent 2° rate constant of 7 × 10 5 M −1s −1. Both the biphasic and monophasic pattern of initial proton liberation observed for myosin and subfragment 1 respectively ( Pemrick, S.M. and F.G. Walz, 1972. J. Biol. Chem. 247 : 2959) can be explained by differences in the relative amounts of the DTNB light chain.