A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking

Abstract

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-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 myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting 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 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 Pi burst method. In Pi 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 kcat of Ca(2+)-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.(ABSTRACT TRUNCATED AT 250 WORDS)

The Small-Molecule Fast Skeletal Troponin Activator, CK-2127107, Improves Exercise Tolerance in a Rat Model of Heart Failure

“…Slow skeletal, fast skeletal, and cardiac myofibrils were prepared from slow bovine masseter, fast rabbit psoas, and bovine heart muscle, respectively, as previously described (Solaro et al, 1971;Young and Davey, 1981;Herrmann et al, 1993). Skeletal isoforms of myosin, actin, troponin, and tropomyosin were purified from rabbit psoas muscle, and cardiac isoforms were purified from the bovine heart, as previously described (Margossian and Lowey, 1982;Pardee and Spudich, 1982;Potter, 1982;Smillie, 1982).…”

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

“…Crosslinking abolishes myofibril shortening in relaxation and contraction making it possible to record polarized intensities. The myofibrils were treated with a water-soluble cross-linker EDC (43)(44)(45). After cross-linking cross-bridges cycle and myofibrils retain normal ATPase activity (46).…”

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies

“…While in myofibrils the cross-bridges bind tightly the ATP before they detach from the thin filament [134], the sequence of the two events in acto-S1 seems to be different [12,104]. The size of the so-called P i -burst indicates that the equilibrium constant of the ATP hydrolysis step is about one order of magnitude higher for myofibrils [52,53,58,89,98] than that reported for unregulated and regulated acto-S1 [50,153]. It is therefore unlikely that the hydrolysis step modulates the rate of ATPase of a muscle as implied by studies of isolated acto-S1.…”