Catalytic cooperativity induced by sulfhydryl1-labeling of myosin filaments

Abstract

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)

Proteasome inhibition improves diaphragm function in congestive heart failure rats

“…The rate constant of tension redevelopment is an estimate for crossbridge cycling kinetics (44), which are slowed in CHF diaphragm (10,52). Impaired cross-bridge cycling kinetics may arise from increased oxidation of contractile proteins (19,35,36,41), but could also be a result of increased myofilament lattice spacing (30,45). Although we cannot exclude that proteasome inhibition affects contractile protein oxidation, our data support the involvement of increased myofilament lattice spacing in CHF diaphragm dysfunction, as proteasome inhibition restored myosin concentration and improved the rate constant of tension redevelopment.…”

Close Proximity of Myosin Loop 3 to Troponin Determined by Triangulation of Resonance Energy Transfer Distance Measurements

“…Cy5-ATP ε 646 = 250000 M −1 cm −1 ; Em λ = 670 nm. 5-TMRIA was prepared based on the method described by Root et al (39) where the fluorophore was dissolved in 1 mL of DMF, added to S1 in a 1:1 molar ratio, and incubated for 30 min. The reaction was terminated by addition of a 10-fold molar excess of DTT and dialyzed in 0.04 M KCl and 10 mM imidazole, pH 7, at 4 °C (TMR ε 543 = 87000 M −1 cm −1 ; Em λ = 567 nm).…”

“…Purified RLC was labeled with AmrB-DTPA via a method previously described by Root (41), but with AmrB used instead of cytosine (AmrB ε 550 = 110000 M −1 cm −1 ; Em λ = 568 nm). Labeled RLC was then exchanged back into S1 by the method of Root et al (39). Absorbance spectra show ~50%, 7%, and 11% efficiency of labeling (not shown) of TMR, FHS, and AmrB, respectively, and an exchange efficiency of RLC-AmrB with endogenous papain S1 RLC of ~5%.…”

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers

“…6). A possible explanation for this difference is that cooperative interactions between active and inactive myosin heads within the myosin filament in muscle fibers compensated for the loss of activity, giving rise to the previously reported less pronounced inactivation in fibers than in solution (22,70). However, inhibition of enzymatic function of myosin can provide only a partial explanation for the inhibition of fiber contractile properties, since reduction of 50 mM H 2 O 2 -oxidized fibers by 100 mM DTT partially restored F max without restoring V 0 or myofibrillar ATPase at pCa 4.5.…”