THE SITE AND STOICHIOMETRY OF THE N-PHENYLMALEIMIDE REACTION WITH MYOSIN WHEN WEAKLY-BINDING CROSSBRIDGES ARE FORMED IN SKINNED RABBIT PSOAS FIBERS

Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N- phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakl y-binding state resembling that of the myosin ATP crossbridge. Under t hese conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358-367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers we re treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [C-14]NPM for 1 h, and homogenize d for SDS-PAGE. 43 +/- 2.2% of the muscle fiber protein ran in the myo sin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 +/- 0.33 moles NPM per mole myosin heavy chain wa s determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major s ites on myosin heavy chain for NPM binding, The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiome try of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain, Comparison of the labeled tryptic peptides with N PM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated Fiber bundles' ATPase activity suggested that the sites for NP M reaction on myosin heavy chain when it locks crossbridges in a weakl y-binding state are Cys-697 (SH2) and Cys-707 (SH1).