The overall goals of our studies are to advance the understanding of the cross-bridge mechanisms in muscular contraction, and to provide insights into the relationships between the biochemical and mechanical events in the cross-bridge cycle. To achieve the major goals chemically treated skinned hamster fiber preparations are utilized having certain moities extracted from the contractile apparatus and then reconstituted with either homologous proteins of the same tissue or the heterologous moities such as from cardiac muscle or from other species. In addition the intact fiber preparation from frog muscle is used in selected experiments to duplicate conditions closer to in vivo. To study properties of the cross-bridge mechanisms, the experiments on control fibers are carried out at various ionic strengths which helps isolate the individual states of cross-bridges that are representative of the configurations during the normal cross-bridge cycle.
The specific aims are to (1) identify the steps in the cycle that are regulated by Ca2+, (2) specify the roles of the regulatory proteins with special emphasis on the identification and characterization of presumably a new cofactor in muscle regulation, and (3) study of the rate limiting steps in the cross-bridge cycle of Vmax and force development. The study is also made of (4) the modification of the cross-bridge kinetics by internal loads that are increased by the compressions of the filament lattice and specifically examines whether the origin of filamentory interactions during such compressions is from particular cross-bridge configurations. The implications of these cross-bridge configurations for the normal contraction and relaxation is examined. In addition (5) the observations on the compartmentalizations of calmodulin in muscle fiber are extended to examine the location of CaM in the fibers by using the immunological techniques combined with EM. The specific roles of the various Ca2+ binding sites in the different aspects of the activation of cross-bridges for contraction are also examined by using different Ca-binding proteins. The results would advance the understanding of the regulatory mechanisms in the contraction process as well as provide new insights into the cross-bridge mechanisms.
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