Revised Manuscript Received April 30, 1993 abstract: In previous work, we studied the early steps of the Mg2+-ATPase activity of Ca2+-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T.(1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrilscontracting with no external load. Here we studied the ATPase of myofibrilscontracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pj burst method. In P; burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the fccat of Ca2+-activated myofibrils was reduced from 1.7 to 0.8 s_1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers. Under relaxing conditions (-Ca2+), the remains large (0.6 s_1) presumably due to rigor activation induced by the 8% heads cross-linked to the thin filaments.

A majorproblem in muscle contraction is to relate the physiological events that occur to the different steps of the Mg2+-ATPase of the myosin heads. Extensive kinetic studies have been carried out on dispersed molecules such as myosin and actomyosin [eg, see Trentham et al.(1976), Taylor (1979), Adelstein and Eisenberg (1980), and Geeves (1991)]. However, in thesesystems the chemical activity is not coupled to mechanical work, and the organized structure of thick and thin filaments is not preserved. The use of skinned fibers allows control of the mechanical behavior, but the limited rate of diffusion of molecules into the center of the fiber prevents the transientkinetic experiments which were so powerful in elucidating the acto-myosin mechanism from being applied.