The regulatory light chains of myosin modulate cross-bridge cycling in skeletal muscle.
We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' -tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling.
Szczesna
D
D
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Zhao
J
J
Potter
J D
JD
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D018994:Myosin Light Chains / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits; D014336:Troponin / Q000302:isolation & purification / Q000378:metabolism; D019209:Troponin C
D003432:Cross-Linking Reagents; D018994:Myosin Light Chains; D014336:Troponin; D019209:Troponin C; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D002118:Calcium; D004229:Dithiothreitol