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A simple and rapid preparation of fully phosphorylated and fully dephosphorylated skeletal muscle myosin. Application to the preparation of a phosphorylated LC2-modified artificial isozyme

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Summary

Fast skeletal myosin LC2 is phosphorylated on ser-15 by a specific myosin light chain kinase (MLCK) in the presence of Ca2+ and calmodulin, and dephosphorylated by a muscle phosphatase in the presence of Mg2+. Fully dephosphorylated myosin is obtained by dialysis of muscle crude extract (0.06m NaCl, 0.01m Tris-HCl, pH 7.5, 50 μm EGTA); fully phosphorylated myosin is obtained by addition of Ca2+ (0.2mm), Mg2+ (10mm) and ATP (3mm) and 5 min incubation at 28° C.

The following reaction characteristics were noted. (1) The crude extract is a very efficient phosphorylating complex and can be diluted to phosphorylate or dephosphorylate purified myosin. (2) Phosphorylation and dephosphorylation appear monophasic, showing no evidence of negative cooperativity in this particular type of myosin and medium. (3) Phosphorylation is 24 times slower in the presence of 0.45m KCl, 5mm pyrophosphate. (4) Thiophosphorylated myosin is slowly dephosphorylated by phosphatase. (5) At the crude myosin stage the dephosphorylation reaction is efficiently inhibited (at 0–4° C) by the presence of 70mm NaF. (6) Myosin-[(T)-LC2′] (a myosin species in which LC2 has been selectively modified by trypsin) is an interesting species refractory to phosphorylation. (7) The myosin-[(T)-LC2′] isozyme can be obtained fully phosphorylated by phosphorylation of myosin followed by limited tryptic proteolysis as described earlier. (8) Urea-PAGE as used separates LC2, phosphoryl-LC2, LC2′ and phosphoryl-LC2′ effectively and in this order.

Through this procedure the (de)-phosphorylating complex isipso facto specific to the myosin species considered; the method avoids lengthy preparations of purified proteins and is easy, rapid and efficient.

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References

  • Bagshaw, C. R. &Kendrick-Jones, J. (1979) Characterization of homologous divalent metal ion binding sites of vertebrate and molluscan myosin using electron paramagnetic resonance spectroscopy.J. molec. Biol. 130, 317–36.

    Google Scholar 

  • Bailey, J. L. (1967) Estimation of Proteins. InTechniques in Protein Chemistry, pp. 340–52. Amsterdam: Elsevier Publishing Company.

    Google Scholar 

  • Bárány, K., Vander Meulen, D. L., Ledvora, R. F. &Bárány, M. (1982) Selective phosphorylation of myosin light chain in intact skeletal muscle.Archs Biochem. Biophys. 217, 392–6.

    Google Scholar 

  • Blumenthal, D. K. &Stull, J. T. (1980) Activation of skeletal muscle myosin light chain kinase by calcium (2+) and calmodulin.Biochemistry 19, 5608–14.

    Google Scholar 

  • Cardinaud, R. (1980) Fate of the light chains in the course of proteolytic digestion of rabbit fast skeletal myosin.Biochimie 62, 135–45.

    Google Scholar 

  • Cardinaud, R. (1982) ‘Artificial’ myosin isozymes; Preparation and characteristics.Eur. J. Biochem. 122, 527–33.

    Google Scholar 

  • Cardinaud, R. &Drifford, M. (1982) Quasi-elastic light scattering studies of rabbit skeletal myosin solutions.J. Musc. Res. Cell Motility 3, 313–32.

    Google Scholar 

  • Cardinaud, R. &Kakol, I. (1985) Influence of the regulatory light chain of fast skeletal muscle myosin on its interaction with actin in the presence and absence of ATP.Biochim. Biophys. Acta 832, 80–8.

    Google Scholar 

  • Chacko, S., Conte, M. A. &Adelstein, R. S. (1976) Phosphorylation of smooth muscle myosin.Fedn Proc. Fedn Am. Socs Exp. Biol. 35, 1581a.

    Google Scholar 

  • Chantler, P. D. &Szent-Györgyi, A. G. (1980) Regulatory light chains and scallop myosin. Full dissociation, reversibility and cooperative effects.J. molec. Biol. 138, 473–92.

    Google Scholar 

  • Gorecka, A., Aksoy, M. O. &Hartshorne, D. J. (1976) The effect of phosphorylation of gizzard myosin on actin activation.Biochem. Biophys. Res. Commun. 71, 325–31.

    Google Scholar 

  • Grand, R. J. A. (1982) The structure and function of light chains. InLife Chemistry Reports, Vol. 1., pp. 105–60. Harwood, Academic Publishers GmbH.

    Google Scholar 

  • Guerriero, V. Jr.,Rouley, D. R. &Means, A. R. (1981) Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme.Cell 27, 449–58.

    Google Scholar 

  • Kemp, B. E., Pearson, R. B. &House, C. (1983) Role of basic residues in the phosphorylation of synthetic peptides by myosin light chain kinase.Proc. natn. Acad. Sci. U.S.A. 83, 7471–5.

    Google Scholar 

  • Kemp, B. E. &Pearson, R. B. (1985) Spatial requirements for location of basic residues in peptide substrates for smooth muscle myosin light chain kinase.J. biol. Chem. 260, 3355–9.

    Google Scholar 

  • Kendrick-Jones, J. &Jakes, R. (1977) Regulatory light chain in myosin. InExcitation-contraction Coupling in Smooth Muscle (edited byCasteels, R., Godfraind, T. andRüegg, J. C.), pp. 343–51. Amsterdam: Elsevier/North Holland Biomedical Press.

    Google Scholar 

  • Khatra, B. S. &Soderling, T. R. (1978) Reversible inhibition of skeletal muscle phosphoprotein phosphatase by ATP, phosphate and fluoride.Biochem. Biophys. Res. Commun. 85, 647–54.

    Google Scholar 

  • Kunz, P. A., Loth, K., Watterson, J. G. &Schaub, M. C. (1980) Nucleotide induced head-head interaction in myosin.J. Musc. Res. Cell Motility 1, 15–30.

    Google Scholar 

  • Michnicka, M., Kasman, K. &Kakol, I. (1982) The binding of actin to phosphorylated and dephosphorylated myosin.Biochim. Biophys. Acta 704, 470–5.

    Google Scholar 

  • Morgan, M., Perry, S. V. &Ottaway, J. (1976) Myosin light chain phosphatase.Biochem. J. 157, 687–97.

    Google Scholar 

  • Mrwa, U. &Hartshorne, D. J. (1980) Phosphorylation of smooth muscle myosin and myosin light chain.Fedn Proc. Fedn Am. Socs Exp. Biol. 39, 1564–8.

    Google Scholar 

  • Nagamoto, H. &Yagi, K. (1984) Properties of myosin light chain kinase prepared from rabbit skeletal muscle by an improved method.J. Biochem. 95, 1119–30.

    Google Scholar 

  • Okamoto, Y. &Yagi, K. (1976) Ca2+-induced conformational changes of spin labeled g2 chain bound to myosin and the effect of phosphorylation.J. Biochem. 80, 111–20.

    Google Scholar 

  • Persechini, A. &Hartshorne, D. J. (1983) Ordered phosphorylation of the two 20 000 molecular weight light chains of smooth muscle myosin.Biochemistry 22, 470–6.

    Google Scholar 

  • Persechini, A. &Stull, J. T. (1984) Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on acto-myosin adenosine triphosphatase activity.Biochemistry 23, 4144–50.

    Google Scholar 

  • Pires, E., Perry, S. V. &Thomas, M. A. W. (1974) Myosin light chain kinase. A new enzyme from striated muscle.FEBS Lett. 41, 292–6.

    Google Scholar 

  • Pires, E. &Perry, S. V. (1977) Purification and properties of myosin-light chain kinase from fast skeletal muscle.Biochem. J. 167 137–46.

    Google Scholar 

  • Sellers, J. R., Chock, P. B. &Adelstein, R. S. (1983) The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state.J. biol. Chem. 258, 14181–8.

    Google Scholar 

  • Sobieszek, A. (1977) Vertebrate smooth muscle myosin. Enzymatic and structural properties. InThe Biochemistry of Smooth Muscle (edited byStephens, N. L.), pp. 413–43), Baltimore: University Park Press.

    Google Scholar 

  • Srivastava, S. &Hartshorne, D. J. (1983) Conversion of a Ca2+-dependendent myosin light chain kinase from skeletal muscle to a Ca2+-independent form.Biochem. Biophys. Res. Commun. 110, 701–8.

    Google Scholar 

  • Stull, J. T., Nunnally, M. H., Moore, R. L. &Blumenthal, D. K. (1985) Myosin light chain kinases and myosin phosphorylation in skeletal muscle. InAdvances in Enzyme Regulation Vol. 23 (edited byWeber, G.), pp. 123–40 Oxford: Pergamon Press.

    Google Scholar 

  • Yasawa, M. &Yagi, K. (1977) A calcium binding subunit of myosin light chain kinase.J. Biochem. 82, 287–9.

    Google Scholar 

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  1. Service de Biophysique, Département de Biologie, Centre d'Etudes Nucleaires de Saclay, F-91191, Gif sur Yvette Cedex, France

    R. Cardinaud

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Cardinaud, R. A simple and rapid preparation of fully phosphorylated and fully dephosphorylated skeletal muscle myosin. Application to the preparation of a phosphorylated LC2-modified artificial isozyme. J Muscle Res Cell Motil 7, 455–466 (1986). https://doi.org/10.1007/BF01753588

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  • DOI: https://doi.org/10.1007/BF01753588

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