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. 1983 Apr 1;96(4):970–978. doi: 10.1083/jcb.96.4.970

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

RL Moss, GG Giulian, ML Greaser
PMCID: PMC2112315  PMID: 6403557

Abstract

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bagshaw C. R., Reed G. H. The significance of the slow dissociation of divalent metal ions from myosin 'regulatory' light chains. FEBS Lett. 1977 Sep 15;81(2):386–390. doi: 10.1016/0014-5793(77)80560-7. [DOI] [PubMed] [Google Scholar]
  2. Chantler P. D., Szent-Györgyi A. G. Regulatory light-chains and scallop myosin. Full dissociation, reversibility and co-operative effects. J Mol Biol. 1980 Apr 15;138(3):473–492. doi: 10.1016/s0022-2836(80)80013-1. [DOI] [PubMed] [Google Scholar]
  3. Greaser M. L., Gergely J. Reconstitution of troponin activity from three protein components. J Biol Chem. 1971 Jul 10;246(13):4226–4233. [PubMed] [Google Scholar]
  4. Guerriero V., Jr, Rowley D. R., Means A. R. Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. Cell. 1981 Dec;27(3 Pt 2):449–458. doi: 10.1016/0092-8674(81)90386-x. [DOI] [PubMed] [Google Scholar]
  5. Julian F. J., Moss R. L. Effects of calcium and ionic strength on shortening velocity and tension development in frog skinned muscle fibres. J Physiol. 1981 Feb;311:179–199. doi: 10.1113/jphysiol.1981.sp013580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Julian F. J., Moss R. L., Waller G. S. Mechanical properties and myosin light chain composition of skinned muscle fibres from adult and new-born rabbits. J Physiol. 1981 Feb;311:201–218. doi: 10.1113/jphysiol.1981.sp013581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Julian F. J. The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J Physiol. 1971 Oct;218(1):117–145. doi: 10.1113/jphysiol.1971.sp009607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kendrick-Jones J., Lehman W., Szent-Györgyi A. G. Regulation in molluscan muscles. J Mol Biol. 1970 Dec 14;54(2):313–326. doi: 10.1016/0022-2836(70)90432-8. [DOI] [PubMed] [Google Scholar]
  9. Kerrick W. G., Hoar P. E., Cassidy P. S., Bolles L., Malencik D. A. Calcium-regulatory mechanisms. Functional classification using skinned fibers. J Gen Physiol. 1981 Feb;77(2):177–190. doi: 10.1085/jgp.77.2.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lehman W. Thick-filament-linked calcium regulation in vertebrate striated muscle. Nature. 1978 Jul 6;274(5666):80–81. doi: 10.1038/274080a0. [DOI] [PubMed] [Google Scholar]
  12. Lowey S., Risby D. Light chains from fast and slow muscle myosins. Nature. 1971 Nov 12;234(5324):81–85. doi: 10.1038/234081a0. [DOI] [PubMed] [Google Scholar]
  13. Léger J. J., Marotte F. The effects of concentrated salt solutions on the structure and the enzymatic activity of myosin molecules from skeletal and cardiac muscles. FEBS Lett. 1975 Mar 15;52(1):17–21. doi: 10.1016/0014-5793(75)80627-2. [DOI] [PubMed] [Google Scholar]
  14. Moss R. L., Giulian G. G., Greaser M. L. Physiological effects accompanying the removal of myosin LC2 from skinned skeletal muscle fibers. J Biol Chem. 1982 Aug 10;257(15):8588–8591. [PubMed] [Google Scholar]
  15. Moss R. L. Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths. J Physiol. 1979 Jul;292:177–192. doi: 10.1113/jphysiol.1979.sp012845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  17. Pemrick S. M. Comparison of the calcium sensitivity of actomyosin from native and L-2-deficient myosin. Biochemistry. 1977 Sep 6;16(18):4047–4054. doi: 10.1021/bi00637a017. [DOI] [PubMed] [Google Scholar]
  18. Sivaramakrishnan M., Burke M. The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full enzymatic activity. J Biol Chem. 1982 Jan 25;257(2):1102–1105. [PubMed] [Google Scholar]
  19. Szent-Györgyi A. G., Szentkiralyi E. M., Kendrick-Jonas J. The light chains of scallop myosin as regulatory subunits. J Mol Biol. 1973 Feb 25;74(2):179–203. doi: 10.1016/0022-2836(73)90106-x. [DOI] [PubMed] [Google Scholar]
  20. Thames M. D., Teichholz L. E., Podolsky R. J. Ionic strength and the contraction kinetics of skinned muscle fibers. J Gen Physiol. 1974 Apr;63(4):509–530. doi: 10.1085/jgp.63.4.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wagner P. D., Giniger E. Hydrolysis of ATP and reversible binding to F-actin by myosin heavy chains free of all light chains. Nature. 1981 Aug 6;292(5823):560–562. doi: 10.1038/292560a0. [DOI] [PubMed] [Google Scholar]
  22. Weeds A. G., Lowey S. Substructure of the myosin molecule. II. The light chains of myosin. J Mol Biol. 1971 Nov 14;61(3):701–725. doi: 10.1016/0022-2836(71)90074-x. [DOI] [PubMed] [Google Scholar]
  23. Wikman-Coffelt J., Srivastava S., Mason D. T. Dissociation and reassociation of rabbit skeletal muscle myosin. Biochimie. 1979;61(11-12):1309–1314. doi: 10.1016/s0300-9084(80)80290-2. [DOI] [PubMed] [Google Scholar]

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