Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.
The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.
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Molecular Biology Institute, University of California, Los Angeles 90024.
Reisler
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D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D027682:Cation Transport Proteins; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D009119:Muscle Contraction; D009126:Muscle Relaxation; D009132:Muscles / Q000502:physiology*; D009210:Myofibrils / Q000502:physiology*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D011817:Rabbits
D000199:Actins; D027682:Cation Transport Proteins; D004228:Dithionitrobenzoic Acid; D000251:Adenosine Triphosphatases; C022761:potassium transporting ATPase; D009218:Myosins