The Strength of Binding of the Weakly-Binding Crossbridge Created by Sulfhydryl Modification has very Low Calcium Sensitivity

Abstract

The acto-subfragment-1*ATP state is an important intermediate in the Ca-activated acto-S1 ATPase reaction, suggesting that the myosin• ATP crossbridge seen in muscle fibers similarly may be an important intermediate in the contractile cycle. Treatment of muscle fibers with either para-phenylenedimaleimide (pPDM) or N-phenylmaleimide (NPM) alters the myosin crossbridges so that they bind to the actin filament with about the same affinity as the myosin•ATP crossbridge. Additionally, the treated crossbridges and the myosin•ATP crossbridge have virtually identical attachment and detachment rate constants. Thus the treated crossbridges appear to be reasonable analogues of the weakly-binding myosin•ATP crossbridges of relaxed fibers and studies of the treated fibers may shed some light on the behavior of the physiologically important myosin•ATP crossbridge. We have examined the influence of Ca²⁺ on the binding and rate constants of pPDM- and NPM-treated weakly-binding crossbridges. In agreement with earlier solution studies, we found almost no Ca-sensitivity of the binding of pPDM- or NPM-treated crossbridges.