Laser photolysis studies of the chemomechanical properties of the cross-bridges in mammalian skeletal muscle

Understanding the mechanism of force generation or shortening in muscle requires knowledge of the relation between biochemical and mechanical steps in the cross-bridge cycle. Indirect evidence from intact muscle fibers indicates that Ca{dollar}sp{lcub}2+{rcub}{dollar} regulates the interaction of actin (A) and myosin (M) by effecting cross-bridge attachment. We tested this hypothesis in permeabilized fibers using adenosine-5{dollar}spprime{dollar}-({dollar}gamma{dollar}-thio) triphosphate (ATP({dollar}gamma{dollar}S)), slowly hydrolyzed ATP analog, and caged ATP({dollar}gamma{dollar}S). With ATP{dollar}lbrackgamma{dollar}S) and Ca{dollar}sp{lcub}2+{rcub}{dollar}, AM.ATP({dollar}gamma{dollar}S) is the predominant cross-bridge state. Our speed-dependent stiffness measurements indicated that AM.ATP({dollar}gamma{dollar}S) is a good analog of weak binding (low force) ATP-cross-bridges. With ATP({dollar}gamma{dollar}S) we observed a Ca{dollar}sp{lcub}2+{rcub}{dollar}-dependent increase of sinusoidal stiffness without force development. The data supports the hypothesis that Ca{dollar}sp{lcub}2+{rcub}{dollar} binding to the thin filament regulatory system can control the attachment of weak binding cross-bridges.; A candidate for the rate limiting step is the dissociation of ADP from AM.ADP. We tested this hypothesis by examining mechanical properties of permeabilized fibers during relaxation or activation, from rigor, initiated by photolysis of caged ATP (absence and presence of Ca{dollar}sp{lcub}2+{rcub}{dollar}) with various steady (MgADP). Our estimated ADP dissociation rate was slow ({dollar}ksb{lcub}rm -D{rcub}{dollar} = 13-35 s{dollar}sp{lcub}-1{rcub}{dollar}) but not rate limiting ({dollar}le{dollar}3 s{dollar}sp{lcub}-1{rcub}{dollar}). By altering the mechanical strain (negative and positive) on the cross-bridges prior to photolysis, we found {dollar}ksb{lcub}rm -D{rcub}{dollar} to be faster for negatively than positively strained cross-bridges. This is consistent with the observed increase in the actomyosin ATPase rate in shortening compared to isometric muscle.; Force generation in muscle has been linked to dissociation of orthophosphate (P{dollar}sb{lcub}rm i{rcub}{dollar}) from AM.ADP.P{dollar}sb{lcub}rm i{rcub}{dollar}. To characterize this relationship, we studied changes in mechanical properties of isometrically active fibers during photolysis of caged P{dollar}sb{lcub}rm i{rcub}{dollar} with various initial (P{dollar}sb{lcub}rm i{rcub}rbrack{dollar}. The rate and amplitude of the resultant tension decline was dependent on the final (P{dollar}sb{lcub}rm i{rcub}{dollar}), with the rate appearing to saturate. To accommodate the data, it was necessary to include a force-generating state, AM{dollar}spprime{dollar}.ADP.P{dollar}sb{lcub}rm i{rcub}{dollar}, such that force generation would occur in a reversible isomerization (AM.ADP.P{dollar}sb{lcub}rm i{rcub} leftrightarrow{dollar} AM{dollar}spprime{dollar}.ADP.P{dollar}sb{lcub}rm i{rcub}{dollar}). We were able to estimate the rate constants for this isomerization and the release of P{dollar}sb{lcub}rm i{rcub}{dollar} at 10 and 20{dollar}spcirc{dollar}C.; Our results indicate that when Ca{dollar}sp{lcub}2+{rcub}{dollar} is present, the active biochemical cycle is initiated allowing myosin to attach to actin in a low-force state (AM.ADP.P{dollar}sb{lcub}rm i{rcub}{dollar}). Force generation occurs during a rapid isomerization of this biochemical state and then P{dollar}sb{lcub}rm i{rcub}{dollar} dissociates. The subsequent strain-dependent dissociation of ADP is slow in isometric muscle and increases with muscle shortening. The cycle continues as ATP rapidly binds to AM, cross-bridges dissociate then hydrolyze ATP and reattach to actin. (Abstract shortened by UMI.)...