Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers

Arata, Toshiaki; Shimizu, Hiroshi

doi:10.1016/0022-2836(81)90004-8

Cited by 27 publications

(9 citation statements)

References 46 publications

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“…Such a conclusion is consistent with other results (6) . Changes in polarization were also observed during activation ofthe fiber, when only part ofmyosin (20-40%) interacts with actin (35,36). However, these changes were quantitatively as well asqualitatively different from those accompanying formation of rigor bonds: p1 remained almost constant and p ii increased, surpassing the values obtained in the rigor state .…”

Section: Discussionmentioning

confidence: 99%

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

Próchniewicz-Nakayama¹,

Yanagida²,

Oosawa³

1983

The Journal of Cell Biology

101

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F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1 :1 molar ratio . Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca" . Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor . The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension . On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution . It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development .Early studies on the properties of skeletal muscle actin suggested that some transformations of the polymer structure upon interaction with myosin are involved in the mechanism of contraction (1) . Later findings that F-actin is a flexible polymer and that its flexibility increases upon binding of myosin heads in vitro as well as in vivo (2-10) and that immobilization of thin filaments in muscle ghost fibers by cross-linking with glutaraldehyde inhibits development of isometric tension (11) supported such a possibility. In this paper we compare directly the conformation of F-actin in a glycerinated muscle fiber in rigor, the relaxed state, and during isometric contraction . As a probe for monitoring conformational changes in F-actin, a phalloidin-fluorescein complex (phalloidin-FITC'), which is a fluorescent derivative of mushroom toxin phalloidin, was used .FITC-labeled phalloidin has been already successfully used for visualization of F-actin bundles in nonmuscle cells (12). We have found that phalloidin-FITC can be bound to F-actin in a glycerinated muscle fiber without affecting either tension or its regulation by Ca 2+ .Since, in skeletal muscle, F-actin filaments are aligned parallel to the axis of the muscle fiber, we could measure the polarized fluorescence from the bound 'Abbreviation used in this paper : FITC, fluorescein isothiocyanate . THE JOURNAL OF CELL BIOLOGY " VOLUME 97 DECEMBER 1983 1663-1667 0 The Rockefeller University Press -0021-9525/83/12/1663/05 $1 .00 REGULAR ARTICLES fluorophore using light polarized either perpendicular or parallel to the axis of the fiber . The different values of polarization obtained in the rigor, relaxed, and active states of the fiber suggest that specific conformational changes in F-actin take place during development of isometric tension . A preliminary report of a portion of these results has appeared (13). MATERIALS AND METHODSMaterials: Glycerinated fibers from rabbit psoas muscle with a sarcomere length from 2 .0 t...

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Section: Discussionmentioning

confidence: 99%

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

Próchniewicz-Nakayama¹,

Yanagida²,

Oosawa³

1983

The Journal of Cell Biology

101

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“…[20][21][22][23] Although in vitro structures are available for most of the troponin components and their complexes, obtained by X-ray, NMR and FRET methods, it is important to observe dynamic interactions in the complexes in situ. Electron paramagnetic resonance (EPR) is a powerful tool for measuring the mobility of spin labels attached to a domain in a protein, [24][25][26][27][28][29][30] by determining the angle of spin label toward a magnetic field [31][32][33][34][35] and the distance between two spin labels. 36,37 The methods for exchange of TnC and myosin light chain in muscle fibres [38][39][40] have made it possible to follow the conformational changes of TnC or myosin neck in permeabilized muscle fibre systems.…”

Section: Introductionmentioning

confidence: 99%

Calcium Structural Transition of Human Cardiac Troponin C in Reconstituted Muscle Fibres as Studied by Site-directed Spin Labelling

Nakamura

Ueki

Hara³

et al. 2005

Journal of Molecular Biology

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“…Various methods have been used in an attempt to define or detect the orientation or the motion of the cross-bridges. They include measurement of dichroism with a dichroic label (Fan, 1963;Fan and Hong, 1964;Fan and Wen, 1979), measurement of the polarization of intrinsic fluorescence (Guth, 1980), measurement of the polarization of fluorescence with a fluorescent probe (Niehei et al, 1974;Borejdo, 1979), measurement of electron paramagnetic resonance with spin labels (Thomas et al, 1975;Thomas, 1978;and Thomas et al, 1979;Thomas and Cooke, 1980;Arata and Shimizu, 1981; Dr. Fan's permanent address is Shanghai Institute of Physiology, Academia Sinica, Shanghai, China.…”

Section: Introductionmentioning

confidence: 99%

The active cross-bridge motions of isolated thick filaments from myosin-regulated muscles detected by quasi-elastic light scattering

Fan¹,

Dewey²,

Colflesh³

et al. 1985

Biophysical Journal

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Intensity fluctuation spectroscopy has been used successfully as a probe that can detect an increase in high-frequency internal motions of isolated thick filaments of Limulus muscle upon the addition of calcium ions. We have attributed such motions to cross-bridge motion instead of to an increase in the flexibility of the filament backbone. Here we show that after cleavage of the S-1 and then the S-2 moieties with papain, cross-linking the myosin heads to the filament backbone, or heat denaturation (42 degrees C, 10 min), the increase in the high frequency internal motions in the thick filaments no longer occurs. Congo Red, which has been shown to induce shortening of isolated myofibrils, also increases the high-frequency motions of the isolated filaments. Furthermore, the increase is suppressed by treating the filaments with a myosin ATPase inhibitor such as vanadate ions (10 mM) or by replacing ATP with either an equimolar CrADP or the nonhydrolyzable ATP analogue beta, gamma-imido-adenine-5'-triphosphate (AMP-PNP). Calcium ions have a similar effect on isolated thick filaments from scallop muscle, where the myosin is known to be regulatory. Calcium ions have no such effect on thick filaments isolated from frog muscle, which is believed not to be regulated by calcium binding to myosin. These results confirm our earlier supposition that the additional high frequency internal motions of the thick filaments isolated from striated muscle of Limulus are related to the energy dependent, active cross-bridge motions.

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Spin-label study of actin-myosin-nucleotide interactions in contracting glycerinated muscle fibers

Cited by 27 publications

References 46 publications

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin.

Calcium Structural Transition of Human Cardiac Troponin C in Reconstituted Muscle Fibres as Studied by Site-directed Spin Labelling

The active cross-bridge motions of isolated thick filaments from myosin-regulated muscles detected by quasi-elastic light scattering

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