Evidence for the presence of a novel thiol group modified together with SH2 in the active site of skeletal myosin ATPase.

Abstract

N-Ethylmaleimide (NEVI), a thiol reagent, specifically modifies SHl and SHE, in the active site of ATPase in skeletal myosin.') Although it is impossible to modify SH~~ alone without modifying SH1, SHl modification with NEM does not affect the essential physiological properties of myosin.'' Accordingly, the physiological function of the local area containing SHE, (SH2 region) has been studied by using SHl-NEM modified myosin (myosin*) or SHrNEM modified subfragment-1 (S-1°) on the structural basis>> as follows. The rate constant of SH modification increases in the presence of Mg2-ATP or in the presence of both Mg-ATP (not Mg-ADP) and F-actin. In addition, physiological level of free Cat concentration regulates the rate of SH. modification in the presence of Mgt-ATP (not Mgt+-ADP).4} >> As an increase in the rate constant of SIL modification means an increase in the conformational change around the SH_,,the aforementioned findings on the molecular level suggest that SH2 region plays a physiologically important function. The present communication shows the evidence that a novel thiol group in S-1° is simultaneously modified together with SH2 in a similar manner to that of the SH.~ modification. Materials and methods. Myosin was prepared from rabbit skeletal back muscle according to the method of Kielley and Bradley. 7) Subfragment-1 was prepared from myosin by chymotryptic digestion.s) Chemical modification of SH1 in subfragment-1 with NEM was performed as reported previously and S-1 was extensively dialyzed against 0.5 M KC1. Chemical modification of SH., and novel thiol group in S-1" was carried out with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM)," a fluorescent maleimide derivative. DACM is insoluble in water and was solubilized in acetone. To protect the active site of ATPase from denaturation due to the organic solvent, care was taken to mako final acetone concentration in the reaction medium not to exceed 2.5%. The number of thiol groups was determined by titration with p-chloromercuribenzoic acid according to the method of Boyer.10 Cat+-ATPase activity was determined in the reaction medium containing 20 mM histidine buffer, pH 7.6, 0.55 M KC1, 5 mM CaCI.~, 1 mM ATP, and 0.1 ml of modified S-a ., solution in a total volume of 1.0 ml. The reaction was carried out at 37°C for 5 min. Inorganic phosphate liberated from ATP was determined according to the method of Fiske and SubbaRow.11> ~) To whom correspondence should be addressed.