The sulfhydryl groups involved in the active site of myosin B adenosinetriphosphatase. VII. A chemical modification of colonic smooth myosin B with N-ethylmaleimide.

The reactivity of the sulfhydryl groups in colonic myosin B to N-ethylmaleimide (NEM) was studied under various conditions. A higher concentration of NEM was required to alter the ATPase activity as compared with skeletal myosin B. A chemical modification of colonic myosin B at a low ionic strength brought about the marked inhibition of EDTA-ATPase and 30-40% activation of Mg2+-ATPase, but had little effect on Ca2+-ATPase. The same results were obtained with NEM modification at a high ionic strength. The presence of 10 mM MgCl2 in the reaction medium for NEM modification caused the activation of Ca2+-ATPase and remarkable inhibition of EDTA-ATPase, suggesting that SH1 is here reactive to NEM. NEM modification of colonic myosin B in the presence of ATP resulted in the inhibition of not only EDTA-ATPase but also Ca2+- and Mg2+-ATPases although the degree of inhibition was different. Addition of 2.0 M urea to the modification medium caused the simultaneous inhibition of EDTA- and Ca2+-ATPase but little change in Mg2+-ATPase. Based on these results, the presence of three specific sulfhydryl groups, SHa, SH1, and SH2, is discussed.