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The rate of inorganic phosphate (Pi) release, and therefore the crossbridge ATPase rate, was determined in permeabilised rat trabeculae. Contraction was elicited by laser-flash photolysis of NPE-caged ATP, and time-resolved Pi release was monitored using MDCC-PBP, a coumarin-labelled phosphate binding protein, which fluoresces upon Pi binding. The ATPase rate during the first turnover of the total crossbridges (assuming 150 μM myosin heads) was 23/s. The rate decreased to a steady state of 4/s after the eighth turnover (0.5–0.6 s after activation). This rate is comparable to published values of 3–10/s, made ∼15 s after activation using a NADH-linked enzyme assay of ADP release. The advantage of using MDCC-PBP is that the control of mechanochemical coupling can be examined from the onset of force production. Force production and Pi release were simulated using a seven-step scheme. Force was attributed to the states in the sequence A.M.ADP.Pi↔A.M.ADP1↔A.M.ADP2, with strain sensitivity incorporated into the isomerisation of A.M.ADP. The A.M.ADP.Pi and A.M.ADP2 states populated rapidly as force was increasing. In contrast, the preisomerisation A.M.ADP1 accumulated slowly after the force plateau was reached and became the dominant force-bearing state at the time of the eighth crossbridge turnover. Experiments are ongoing to examine how the distribution of A.M states changes in response to rapid length changes.
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