The Sulfhydryl Groups Involved in the Active Site of Myosin B Adenosinetriphosphatase

II. Effect of Modification of the S_a Thiol Group on Superprecipitation and Clearing

Abstract

The effect of S_a modification with NEM, which activates Mg²⁺-ATPase through an enhancement of the association of actin and myosin, was investigated on the superprecipitation, clearing and Mg²⁺-ITPase of myosin B with reference to the effect of S₁-blocking.
1. Superprecipitation induced by ATP was markedly enhanced by S_a-blocking even at high concentrations of Mg²⁺ and substrate; this may be due to an increase in the affinity of myosin and actin on blocking S_a.
2. Nevertheless, neither ITP-induced superprecipitation nor Mg²⁺-ITPase was affected by S_a modification.
3. Blocking of S₁ brought about the inhibition of ATP- and ITP-induced superprecipitation and Mg²⁺-ITPase activity, suggesting that S_a-blocking decreases the affinity of myosin and actin.
4. S_a-blocked myosin B showed greater resistance to clearing by ATP, especially in the presence of Ca²⁺ ions, whereas in the clearing response of actomyosin gel to PP₁ no difference between S_a-blocked and unmodified myosins B was observed. On the other hand, the clearing response of myosin B became more sensitive to both ATP and PP₁ on blocking S₁.
Based on the above results and preliminary data suggesting that S_a is located in LMM, the interaction of myosin filaments and actin filaments under physiological conditions is discussed.