Regulation of Tension and Active State in the Heart

Pemrick, Suzanne

Abstract

Both tension and active state in the heart must ultimately be explained at the molecular level in terms of the steady state turnover of actomyosin complexes. This investigator has begun to describe a regulatory function for the L2 light chain (LC) of vertebrate-striated muscle. L2 increases the steady state concentration of A-M**ADP.P complexes and the apparent CA ion-affinity of troponin. Phosphorylation of L2 (L2P) decreases the Kapp of the actomyosin and acto-HMM ATPase. The mechanism of L2 function will be studied in the cardiac and skeletal system. A functional analysis and purification of the physiologically relevant LC kinase will determine whether L2P is a unicomponent modifier. Steady state kinetic analysis of the acto-HMM ATPase as a function of L2 and L2P content will be correlated with binding studies (turbidity, ultracentrifugation, and fluorescently labelled F-actin). These data will indicate whether L2 regulates the rate of formation or the rate of breakdown of A-M**ADP.P complexes. The ability of L2 and L2P to induce a conformational change in myosin will be analyzed by a time-course profile on sodium dodecyl sulfate polyacrylamide gels of the products of digestion by alpha-chymotrypsin. This approach will produce a methodology for preparing HMM with 2 mol of undegraded and associated L2/mol. Affinity chromatography, radioactivity assays, and electrophoresis in nondenaturing systems of L2-deficient myosin, will determine if striated myosin contains two functionally distinct populations of L2. Recently, this laboratory has observed that the Ca ion-activated neutral proteases from platelets and dystrophic skeletal myosin degrades the heavy chain of unphosphorylated, but not phosphorylated myosin. The SH1 thiol of unphosphorylated myosin will be alkylated with a radioactive reagent, and following digestion by this protease, the heavy chain fragment will be analyzed for radioactivity. This approach will determine whether L2 is conformationally linked to the active site of myosin. The correlation between active tension and L2P indicates the importance of the observation that L2P renders myosin resistant to degradation by Ca ion-activated proteases.