The mechanism by which muscle converts chemical energy in the form of adenosine triphosphate (ATP) into mechanical energy as work, force and shortening (chemomechanical transduction) will be studied. Current theories of chemomechanical transduction are based on kinetic analyses of the ATPase of isolated muscle proteins in solution and mechanical measurements on muscles cells. These theories require that the rate of ATP hydrolysis is regulated by how fast the muscle shortens or lengthens and assume that the rate at which ATP hydrolysis products, inorganic phosphate (Pi) and adesoine diphosphate (ADP), are released by the crossbridges is determined by the the force or displacement applied to the crossbridges. The rate of ATP is hydrolysis by shortening or lengthening mammalian muscles and whether force or displacement affects the rate of product release by the crossbridge are not known. The development of 2-nitro benzyl derivates of ATP, ADP, and Pi, which can rapidly (greater than 100 s-1) photogenerate ATP, ADP, or Pi allows measurement of the rate of ATP hydrolysis and, by employing transient kinetic techniques, the rate constants for Pi and ADP release and binding in single glycerinated muscle fibers. The studies described in this application are designed to experimentally measure the influence velocity of shortening and lengthening have on the rate of ATP hydrolysis, the efficiency of work performance, and product release rate constants in single glycerinated muscle fibers. In theories of contractile regulation, the force development and actomyosin ATPase rate are controlled by the sarcoplasmic calcium concentration by either a steric blocking mechanism or modulation of the rate of Pi release from the actomyosin-products complex. Both mechanisms predict that the isometric force and ATPase rate should rise in parallel with increases in the sarcoplasmic calcium concentration. This hypothesis, as well as the effect of calcium concentration on the rate of Pi release from the crossbridge, will be examined in glycerinated muscle fibers are proposed in which the minimum distance a crossbridge can remain attached to the thin. These simultaneous measurements of mechanical behavior and chemical change in a system whose structural integrity and mechanical restrains are preserved and under direct experimental control, permits rigorous testing of current theories of contraction.
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