A STRUCTURAL AND KINETIC-STUDY ON MYOFIBRILS PREVENTED FROM SHORTENING BY CHEMICAL CROSS-LINKING

In previous work, we studied the early steps of the Mg2+-ATPase activi ty of Ca2+-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T.(1992) Biochemistry 31, 1564-1569]. The myofibrils were free to con tract, and the results obtained refer to the ATPase cycle of myofibril s contracting with no external load. Here we studied the ATPase of myo fibrils contracting isometrically. To prevent shortening, we cross-lin ked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). S DS-PAGE and Western blot analyses showed that the myosin rods were ext ensively cross-linked and that 8% of the myosin heads were cross-linke d to the thin filament. The transient kinetics of the cross-linked myo fibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4-degrees- 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 P(i) burst method. In P(i) burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concent rations (as determined by the cold ATP chase methods) for both cross-l inked (isometric) and un-cross-linked (isotonic) myofibrils. This show s that in both cases the rate-limiting step is after the cleavage of A TP. When cross-linked, the k(cat) of Ca2+-activated myofibrils was red uced 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 k(cat) r emains large (0.6 s-1) presumably due to rigor activation induced by t he 8% heads cross-linked to the thin filaments.