Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers

Borejdo, Julian; Assulin, Olga

doi:10.1021/bi00562a033

Cited by 16 publications

(5 citation statements)

References 21 publications

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“…Therefore, we measured the total number of accessible S-1 actin-binding sites in the absence of Ca 2ϩ and ATP at physiological ionic strength. We show that the S-1 binding stoichiometry within cardiac RTFs has similar numbers of binding sites obtained when heavy meromyosin binds to skeletal thin filaments (20) and myofibrils (21). Fig.…”

Section: Discussionsupporting

confidence: 66%

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Dweck¹,

Reynaldo

Pinto³

et al. 2010

Journal of Biological Chemistry

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In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac troponin C (CTnC) associated with dilated cardiomyopathy reduces the Ca 2؉ -activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the calcium affinity of CTnC in troponin complex, regulated thin filaments (RTF), and the Ca 2؉ sensitivity of contraction and ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/ATPase may be due to a defect in the interactions between CTnC and cardiac troponin T, which are known to be necessary for ATPase activation. An additional mechanism for the reduction in force/ATPase comes from measurements of the binding stoichiometry of myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by ϳ23% to 3.7. Altogether, these results suggest that the reduction in force and ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a dilated cardiomyopathy-causing mutation.

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Section: Discussionsupporting

confidence: 66%

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Dweck¹,

Reynaldo

Pinto³

et al. 2010

Journal of Biological Chemistry

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“…Using these fluorescent probes, we have demonstrated several features of $1 binding to actin sites in the myofihril including the calcium sensitivity of the location of binding, and the concentration dependency and cooperativity of binding in the absence of calcium. The binding of fluorescent conjugates of the myosin head to myofibrils has been studied by several researchers (Aronson, 1965;Borejdo and Assulin, 1980;Herman and Pollard, 1978;Sanger, 1975;Sanger et al, 1984). None, however, demonstrated the calcium sensitivity of the location of binding.…”

Section: Discussionmentioning

confidence: 99%

“…The binding of fluorescent conjugates of the myosin head to myofibrils has been studied by several researchers (Aronson, 1965;Borejdo and Assulin, 1980;Herman and Pollard, 1978;Sanger, 1975;Sanger et al, 1984). None, however, demonstrated the calcium sensitivity of the location of binding.…”

Section: Discussionmentioning

confidence: 99%

Regulation of binding of subfragment 1 in isolated rigor myofibrils.

Swartz

Greaser

Marsh

1990

The Journal of Cell Biology

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Abstract.A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin-to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z-lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing IS1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over >2-3 tropomyosintroponin-7 actin functional units. MUSC LE contracts by a sliding of the actin-containing thin filaments over the myosin-containing thick filaments (Huxley and Neidergerke, 1954;Huxley and Hanson, 1954), a process using the ATP driven, cyclical interaction between actin and the myosin head to produce both movement and force. The regulation of contraction in vertebrate skeletal muscle is a complex process employing a minimum of six different protein species and calcium (Ebashi et al., 1969;Weber and Murray, 1973). Both tropomyosin and troponin are required for demonstrating calcium sensitivity of ATPase activity in acto-myosin preparations. The tropomyosin-troponin complex spans seven actin monomers, and this group has been defined as the functional unit of the thin filament . The troponin complex is composed of three subunits, troponin-T, troponin-I, and troponin-C. The latter confers calcium sensitivity to regulated acto-myosin ATPase activity, while the combination of troponin-T and troponin-I without troponin-C inhibits ATPase activity (Greaser and Gergely, 1973).The structural mechanism by which tropomyosin and tropouin regulate contraction has been studied by x-ray diffraction of intact muscle fibers. Early studies demonstrated that there was a difference in the intensity pattern of the actin layer lines between relaxed and contracted muscles; it was explained by a small movement of tropomyosin from a peripheral position on the thin filament to a position nearer to the groove of the actin filament (Haselgrove, 1972;Huxley, 1972;Parry and Squire, 1973). These structural observations and the proposed mechanism for the regu...

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“…For this reason a significant effort has been invested in characterizing the structural and biochemical properties of the complex. A number of studies have shown that maximal saturation of F-actin by SI or HMM was one head per one actin (Young, 1967;Margossian & Lowey, 1978; White & Taylor, 1976;Borejdo & Assulin, 1980; Lehrer & Ishii, 1988). This conclusion was reached by analyzing data obtained at high degrees of saturation of actin filaments with myosin heads.…”

mentioning

confidence: 99%

Two different rigor complexes of myosin subfragment 1 and actin

Oa¹,

Al²,

Vs³

et al. 1993

Biochemistry

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Our previous titration and cross-linking experiments showed that myosin subfragment 1 (S1) can bind to one or two monomers in F-actin [Andreev, O. A., & Borejdo, J. (1991) Biochem. Biophys. Res. Commun. 177, 350-356; (1992a) J. Muscle Res. Cell Motil. 13, 523-533; (1992b) Biochem. Biophys. Res. Commun. 188, 94-101]. In the present work we used a sedimentation method to extend these studies to equilibrium binding and a stopped flow method to investigate its kinetics. Both equilibrium and kinetic data indicated the existence of two different rigor complexes. On the basis of these data we developed a model which suggested that binding of S1 to F-actin occurred in two steps: (i) initial rapid binding to one monomer of F-actin, A + M<==>A.M and (ii) a consequent slow binding to a neighboring monomer, A.M + A<==>A.M.A, where A stands for actin and M for myosin subfragment 1. The second reaction can proceed only if the neighboring actin site is unoccupied. The model fit the equilibrium and kinetic binding data with equilibrium constants K1 = 6 x 10(6) M-1 and K2 = 4 and kinetic constants k+1 = 10.5 x 10(6) M-1 s-1, k-1 = 1.75 s-1, k+2 = 0.8 s-1, and k-2 = 0.2 s-1, where the subscripts refer to the reactions i and ii. These results corroborate our hypothesis that myosin head can make two types of complexes with F-actin and support our speculation that during a power stroke in contracting muscle a myosin head may first bind to one and then to two actins.

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Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers

Cited by 16 publications

References 21 publications

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Regulation of binding of subfragment 1 in isolated rigor myofibrils.

Two different rigor complexes of myosin subfragment 1 and actin

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