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Nucleotides Induced Changes in Skeletal Muscle Myosin by DSC, TMDSC and EPR

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Abstract

Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum.

In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region The monoton decreasing TMDSC heat capacities show that C p of biological samples should not be temperature independent.

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Authors and Affiliations

  1. Department of Biophysics, Faculty of Medicine, University of Pécs, Szigeti út 12, H-7624, Pécs, Hungary

    D. Lőrinczy

  2. Institute of Forensic Medicine, Faculty of Medicine, University of Pécs, Szigeti út 12, H-7624, Pécs, Hungary

    F. Könczöl

  3. Department of Urology, Faculty of Medicine, University of Pécs, Szigeti út 12, H-7624, Pécs, Hungary

    L. Farkas

  4. Faculty of Medicine, Central Research Laboratory of University Pécs, Szigeti út 12, H-7624, Pécs, Hungary

    J. Belagyi

  5. Department of Physics, University of Rostock, Universitätplatz 3, D-18051, Rostock, Germany

    C. Schick

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  1. D. Lőrinczy
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  2. F. Könczöl
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  3. L. Farkas
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  4. J. Belagyi
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  5. C. Schick
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Lőrinczy, D., Könczöl, F., Farkas, L. et al. Nucleotides Induced Changes in Skeletal Muscle Myosin by DSC, TMDSC and EPR. Journal of Thermal Analysis and Calorimetry 66, 633–644 (2001). https://doi.org/10.1023/A:1013197824826

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