In cardiac and skeletal muscles the phosphorylatable or P-light chain of myosin is phosphorylated by a Ca2+ and calmodulin-dependent myosin light chain kinase. In skeletal muscle phosphorylation of P-light chain has been correlated to potentiation of isometric twitch tension. The long-range goals of the research described in this application are to determine the role of myosin P-light chain phosphorylation in the regulation of cardiac and skeletal muscle contractions. The relationship between P-light chain phosphorylation and myosin ATPase activity will be examined with heavy meromyosin, myosin filaments, myofibrils and skinned fibers from cardiac and skeletal muscles. The effect of P-light chain phosphorylation on the mechanical properties (force, velocity) of skinned and non-skinned fibers will be examined. Calmodulin-independent fragments of myosin light chain kinase will be used for phosphorylation, while synthetic peptide inhibitors and monoclonal antibodies will be used to inhibit phosphorylation specifically. The biochemical and mechanical responses will be reversed with phosphoprotein phosphatases. A reversible hypermeable treatment will be used to introduce these proteins and peptides into non-skinned fibers (single cells or small bundles) to affect P-light chain phosphorylation. The biochemical properties of myosin light chain kinase isozymes from skeletal and cardiac muscles will be investigated. Functional domains within kinase molecule will be probed by limited proteolysis and identification of specific peptides by the binding of calmodulin, ATP analogs and monoclonal antibodies. The catalytic properties of purified myosin light chain kinases will be analyzed with synthetic peptide substrates to define the amino acid determinants in the primary sequence that account for marked substrate specificity of the kinases. The physical and kinetic properties of myosin light chain kinase interactions with selected synthetic peptide substrates will also be examined with high resolution nuclear magnetic resonance. These investigations will provide information on the biochemical properties of myosin light chain kinases that will be related to information on other calmodulin-dependent enzymes and other protein kinases. These physiological and biochemical investigations on the regulation of muscle contractions may provide insights into pathophysiological processes associated with derangements of heart and skeletal muscle performance.
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