A structural and kinetic study on myofibrils prevented from shortening by chemical cross-linking.
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)
Herrmann
C
C
INSERM U128, CNRS, BP 5051, Montpellier, France.
Sleep
J
J
Chaussepied
P
P
Travers
F
F
Barman
T
T
D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D003432:Cross-Linking Reagents; D005022:Ethyldimethylaminopropyl Carbodiimide; D066298:In Vitro Techniques; D007537:Isometric Contraction; D007700:Kinetics; D009126:Muscle Relaxation; D009132:Muscles / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits
D000199:Actins; D003432:Cross-Linking Reagents; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide