The goal of this research is to relate physiological, structural and biochemical theories of muscle contraction by measurement of protein motions and biochemical rate constants of the actomyosin ATPase in physiologically active skeletal and heart muscle preparations. Protein structural changes will be detected in electron micrographs of muscle fibers ultra-rapidly frozen during the onset of tension development after activation by laser pulse photolysis of caged ATP or caged Ca2+. Caged molecules are inert, but photolabile precursors of substrate or signaling molecules that release the active compound on irradiation by a pulse of near-u.v. laser light. Protein rotational motions will be measured using a novel time-resolved fluorescence polarization method that will allow microscopic spatial resolution. Fluorescent actin and myosin light chains will be micro-injected into live, isolated cardiac or cultured skeletal myocytes. The orientation of the labeled actin or myosin light chains will be monitored during the time course of spontaneous or electrically stimulated contractions. Specific reaction steps of the ATPase mechanism will be investigated in working muscle fibers by measuring exchange of stable oxygen isotopes between the solvent and phosphate groups in the substrate and products of the ATPase reaction. The distribution of isotopes will be determined by gas chromatography coupled to mass spectrometry of the phosphate compounds. This oxygen exchange techniques probes the elementary biochemical reactions in the intact filament lattice. The influence of filament sliding on these reaction steps will be determined. Results from this project should significantly advance knowledge of the contractile process and thus bring a greater understanding of both normal and pathological states of skeletal and heart muscle and other types of cell mortality.
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