Catalytic cooperativity induced by SH1 labeling of myosin filaments.
Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)
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Molecular Biology Institute, University of California, Los Angeles 90024.
Cheung
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Reisler
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D000199:Actins / Q000378:metabolism; D000205:Actomyosin / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000199:Actins; D046911:Macromolecular Substances; D008301:Maleimides; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D004533:Egtazic Acid; D000205:Actomyosin; D004228:Dithionitrobenzoic Acid; C028525:N-phenylmaleimide; D000251:Adenosine Triphosphatases; C029321:calcium potassium ATPase; D009218:Myosins; D005033:Ethylmaleimide