The generally accepted mechanism of muscle contraction, the sliding filament theory, involves the interaction of two proteins: actin and myosin. These proteins, which exist in vivo as filaments, are arranged in a highly ordered geometrical pattern in which the myosin and actin filaments interdigitate. According to the sliding filament theory projections, which extend from the myosin filament, cyclically interact with the actin filament. Net sliding or contraction occurs by coupling the hydrolysis of ATP to conformational changes in the acto-myosin complexes. In order to gain an understanding of the actin-myosin interaction from a molecular point of view, biochemists have turned to the study of the actin activated myosin subfragment ATPase activity which is believed to be the in vitro correlate to the contraction process. The primary objective of the proposed research is to improve the understanding of the kinetics of the ATP hydrolysis reaction of both cardiac and skeletal muscle myosin. During the proposed research period only the soluble subfragments of myosin, Subfragment-1 (S-1) and Heavy Meromyosin (HMM) will be studied. This work should improve the understanding of how the complete myosin molecule functions in vivo, and may lead to insight into why myosin has two heads. One advantage of a detailed kinetic scheme of the actin activated myosin ATPase is that it may lead to a better understanding of how pathological and altered physiological states affect the ability of muscle to contract. Several laboratories have reported that abnormalities of cardiac muscle can be associated with altered ATPase activities. The changes seen are small and more work is needed to establish their cause and effect relationship. Presteady state kinetics should be more sensitive to these changes. In order to form as complete a kinetic description as is currently possible, it is necessary to perform several steady state and presteady state measurements. These include: steady state and presteady state measurements of the binding of Actin to the soluble myosin subfragments during ATP hydrolysis using stopped flow and airfuge techniques; the rate and magnitude of the initial phosphate burst using quench flow and stopped flow techniques; steady state measurements of the ATPase activity using direct Pi assays or PH stat techniques; and steady state 018 exchange measurements using spectrometric analysis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Modified Research Career Development Award (K04)
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Pathobiochemistry Study Section (PBC)
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State University New York Stony Brook
Schools of Medicine
Stony Brook
United States
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