IN FY92, our study of the myosin crossbridge interaction with actin in muscle fibers focused mainly on the weakly-binding crossbridge interaction. We examined the changes that occur when alkylating agents such as N-phenyl maleimide (NPM) and para-phenylene diamaleimide (pPDM) bind to muscle fibers and lock the crossbridges in a weakly-binding configuration. We observed that titin and myosin are the two major proteins that NPM and pPDM bind to. The binding of NPM to titin does not appear to have much effect, but binding accompanied by crosslinking of titin by pPDM causes an almost two-fold increase in fiber resting tension. The remaining effects of these agents presumably are due to binding to myosin heavy chain. To support this argument, we have initiated a study designed to measure the stoichiometry of the binding to myosin heavy chain and also to determine the specific binding sites on myosin heavy chain. (These will likely be the SH1 and SH2 reactive sulfhydryls.) Initial results on measuring the stoichiometry showed the efficacy of our techniques, but also suggest that the method we had used satisfactorily for making the sarcolemmas of single fibers permeable, is not adequate for the larger diameter bundles of fibers used in the stoichiometry determination.
