Actin-attached and detached crossbridges in myofibrils: Segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain

Assulin, Olga; Borejdo, Julian; Flynn, Carroll

doi:10.1007/bf01753418

Cited by 8 publications

(3 citation statements)

References 27 publications

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“…Attachment of myosin to actin also decreases the susceptibility to proteolysis of a region of the myosin head in the vicinity of residues 640-650. This susceptibility is partially restored in the presence of MgPPi or MgAMPPNP, with estimates of the fraction of heads dissociated in rough agreement with those found here and by Fajer et al (Chen and Reisler, 1984;Assulin et al, 1986). MgPP; binds strongly to myosin (Kd 0.5 ,tM) but only weakly to the actomyosin complex, Kd 2-4 mM.…”

Section: Discussionsupporting

confidence: 91%

“…These changes could be due to either an actomyosin bond with altered mechanical properties or to cross-bridge detachment, or to both effects. The possibility of cross-bridge detachment has received support from combined stiffness and x-ray diffraction measurements in fibers (Brenner et al, 1986b) as well as proteolytic digestion experiments on myofibrils (Chen and Reisler, 1984;Assulin et al, 1986). Other studies using intact fibers, x-ray diffraction, and electron microscopy have clearly shown that MgAMPPNP produces changes in cross-bridge structure upon binding (Goody, et al, 1976;Lymm, 1975;Marston et al, 1976;Padron and Huxley, 1984;Reedy et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

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Energetics of the actomyosin bond in the filament array of muscle fibers

Pate

Cooke

1988

Biophysical Journal

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The interaction between actin and myosin in the filament array of glycerinated muscle fibers has been monitored using paramagnetic probes and mechanical measurements. Both fiber stiffness and the spectra of probes bound to a reactive sulfydral on the myosin head were measured as the actomyosin bond was weakened by addition of magnesium pyrophosphate (MgPPi) and glycerol. In the absence of MgPPi, all myosin heads are attached to actin with oriented probes. When fibers were incubated in buffers containing MgPPi, a fraction of the probes became disordered, and this effect was greater in the presence of glycerol. To determine whether the heads with disordered probes were detached from actin, spin-labeled myosin subfragment-1 (MSL-S1) was diffused into unlabeled fibers, and the fractions bound to actin and free in the medium were correlated with the oriented and disordered spectral components. These experiments showed that the label was oriented when MSL-S1 was attached to actin in a ternary complex with the ligand and that all heads with disordered probes were detached from actin. Thus the fraction of oriented labels could be used to determine the fraction of heads attached to actin in a fiber in the presence of ligand. The fraction of myosin heads attached to actin decreased with increasing [MgPPi], and in the absence of glycerol approximately 50% of the myosin heads were dissociated at 3.3 mM ligand with little change in fiber stiffness. In the presence of 37% glycerol plus ligand, up to 80% of the heads could be detached with a 50% decrease in fiber stiffness. The data indicate that there are two populations of myosin heads in the fiber. All the data could be fit with a model in which one population of myosin heads (comprising approximately 50% of the total) sees an apparent actin concentration of 0.1 mM and can be released from actin with little change in fiber stiffness. A second population of myosin heads (approximately 50%) sees a higher actin concentration (5 mM) and is only released in the presence of both glycerol and ligand.

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Section: Discussionsupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

Energetics of the actomyosin bond in the filament array of muscle fibers

Pate

Cooke

1988

Biophysical Journal

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“…Conformational changes in the actin filament due to the binding of S-l have been reported by Ando (1989). Evidence that not all myosin heads are optimally activated in myosin filaments comes from a lower Kmax for myosin filaments than for S-l (Pope et al, 1981 fibers also indicate at least two populations of myosin heads seeing different effective actin concentrations (Assulin et al, 1986; Pate & Cooke, 1988;Fajer et al, 1988). These results suggest that the mechanism of increasing myosin activity in the filament may involve at least two processes: recruitment of myosin heads which are inaccessible to the actin filament and increased activation of myosin heads which are already exposed to actin.…”

Section: Discussionmentioning

confidence: 77%

Catalytic cooperativity induced by sulfhydryl1-labeling of myosin filaments

Root

Cheung²,

Reisler³

1991

Biochemistry

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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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Fluorescence polarization study of the rigor complexes formed at different degrees of saturation of actin filaments with myosin subfragment-1

Andreev

Takashi

Borejdo

1995

J Muscle Res Cell Motil

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A serine residue located in the active site of myosin head (S1) was labelled by 9-anthroylnitrile, an amino group located in the central domain of S1 was labelled by 7-diethylamino-3-(4'-isothio-cyanato-phenyl)-4-methylcoumari n, a cysteine residue located near the C-terminus of S1 was labelled by 5-[2-((iodoacetyl)-amino)ethyl]-amino-naphthalene-1-sulfonic acid (1,5-IAEDANS) and a cysteine residue located near the C-terminus of the alkali light chain 1 was labelled with iodoacetamido-tetramethyl-rhodamine. Polarization of fluorescence of S1 was measured in solution (where it indicated the mobility of actin-bound S1) and in myofibrils (where it indicated orientation of probes) to check whether the anisotropy of S1 labelled at different positions depended on the molar ratio S1:actin. In solution, when increasing amounts of actin were added to a fixed amount of labelled S1 (i.e. when myosin heads were initially in excess over actin), anisotropy saturated at 1 mol of S1 per 1 mol of actin. When increasing amounts of S1 were added to a fixed amount of F-actin (i.e. when actin was initially in excess over S1), the anisotropy saturated at 1 mol of S1 per 2 mols of actin. In myofibrils, orientation of S1 was different when S1 was added at nanomolar concentration (intrinsic actin was in excess over extrinsic S1) then when it was added at micromolar concentration (excess of S1 over actin). The fact that the anisotropy of S1 labelled at different positions depended on the molar ratio excluded the possibility that changes were confined to one part of the cross-bridge and supports our earlier proposal that the two rigor complexes which S1 can form with F-actin differ globally in conformation.

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Actin-attached and detached crossbridges in myofibrils: Segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain

Cited by 8 publications

References 27 publications

Energetics of the actomyosin bond in the filament array of muscle fibers

Energetics of the actomyosin bond in the filament array of muscle fibers

Catalytic cooperativity induced by sulfhydryl1-labeling of myosin filaments

Fluorescence polarization study of the rigor complexes formed at different degrees of saturation of actin filaments with myosin subfragment-1

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