Skip to main content
NCBI home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1990 Mar 1;95(3):477–498. doi: 10.1085/jgp.95.3.477

Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers

PMCID: PMC2216322  PMID: 2324702

Abstract

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO4(2-) as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC2 resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca2+] by as much as 0.2 pCa units after LC2 extraction. At low concentrations of Ca2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC2 to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC2. LC2 extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Alexis M. N., Gratzer W. B. Interaction of skeletal myosin light chains with calcium ions. Biochemistry. 1978 Jun 13;17(12):2319–2325. doi: 10.1021/bi00605a010. [DOI] [PubMed] [Google Scholar]
  2. Allen J. D., Moss R. L. Factors influencing the ascending limb of the sarcomere length-tension relationship in rabbit skinned muscle fibres. J Physiol. 1987 Sep;390:119–136. doi: 10.1113/jphysiol.1987.sp016689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bremel R. D., Weber A. Cooperation within actin filament in vertebrate skeletal muscle. Nat New Biol. 1972 Jul 26;238(82):97–101. doi: 10.1038/newbio238097a0. [DOI] [PubMed] [Google Scholar]
  4. Bressler B. H. Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C. Can J Physiol Pharmacol. 1981 Jun;59(6):548–554. doi: 10.1139/y81-082. [DOI] [PubMed] [Google Scholar]
  5. Cornish-Bowden A., Koshland D. E., Jr Diagnostic uses of the Hill (Logit and Nernst) plots. J Mol Biol. 1975 Jun 25;95(2):201–212. doi: 10.1016/0022-2836(75)90390-3. [DOI] [PubMed] [Google Scholar]
  6. Edman K. A. The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres. J Physiol. 1979 Jun;291:143–159. doi: 10.1113/jphysiol.1979.sp012804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  8. Ford L. E., Huxley A. F., Simmons R. M. Tension responses to sudden length change in stimulated frog muscle fibres near slack length. J Physiol. 1977 Jul;269(2):441–515. doi: 10.1113/jphysiol.1977.sp011911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Giulian G. G., Moss R. L., Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem. 1983 Mar;129(2):277–287. doi: 10.1016/0003-2697(83)90551-1. [DOI] [PubMed] [Google Scholar]
  10. Godt R. E., Lindley B. D. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol. 1982 Aug;80(2):279–297. doi: 10.1085/jgp.80.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Godt R. E., Maughan D. W. Influence of osmotic compression on calcium activation and tension in skinned muscle fibers of the rabbit. Pflugers Arch. 1981 Oct;391(4):334–337. doi: 10.1007/BF00581519. [DOI] [PubMed] [Google Scholar]
  12. Goldman Y. E., McCray J. A., Ranatunga K. W. Transient tension changes initiated by laser temperature jumps in rabbit psoas muscle fibres. J Physiol. 1987 Nov;392:71–95. doi: 10.1113/jphysiol.1987.sp016770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Greaser M. L., Moss R. L., Reiser P. J. Variations in contractile properties of rabbit single muscle fibres in relation to troponin T isoforms and myosin light chains. J Physiol. 1988 Dec;406:85–98. doi: 10.1113/jphysiol.1988.sp017370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Güth K., Potter J. D. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J Biol Chem. 1987 Oct 5;262(28):13627–13635. [PubMed] [Google Scholar]
  16. HUXLEY A. F. Muscle structure and theories of contraction. Prog Biophys Biophys Chem. 1957;7:255–318. [PubMed] [Google Scholar]
  17. Holroyde M. J., Potter J. D., Solaro R. J. The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins. J Biol Chem. 1979 Jul 25;254(14):6478–6482. [PubMed] [Google Scholar]
  18. Kössler F., Lange F., Küchler G. Isometric twitch and tetanic contraction of frog skeletal muscles at temperatures between 0 to 30 degrees C. Biomed Biochim Acta. 1987;46(11):809–813. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Margossian S. S., Bhan A. K., Slayter H. S. Role of the regulatory light chains in skeletal muscle actomyosin ATPase and in minifilament formation. J Biol Chem. 1983 Nov 10;258(21):13359–13369. [PubMed] [Google Scholar]
  22. Metzger J. M., Greaser M. L., Moss R. L. Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle. J Gen Physiol. 1989 May;93(5):855–883. doi: 10.1085/jgp.93.5.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Metzger J. M., Moss R. L. Thin filament regulation of shortening velocity in rat skinned skeletal muscle: effects of osmotic compression. J Physiol. 1988 Apr;398:165–175. doi: 10.1113/jphysiol.1988.sp017036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miller L., Reisler E. Light chain dependent effects of actin binding on the S-1/S-2 swivel in myosin. J Mol Biol. 1985 Mar 20;182(2):271–279. doi: 10.1016/0022-2836(85)90345-6. [DOI] [PubMed] [Google Scholar]
  25. Morimoto K., Harrington W. F. Evidence for structural changes in vertebrate thick filaments induced by calcium. J Mol Biol. 1974 Sep 25;88(3):693–709. doi: 10.1016/0022-2836(74)90417-3. [DOI] [PubMed] [Google Scholar]
  26. Moss R. L., Allen J. D., Greaser M. L. Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament. J Gen Physiol. 1986 May;87(5):761–774. doi: 10.1085/jgp.87.5.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moss R. L. Effects on shortening velocity of rabbit skeletal muscle due to variations in the level of thin-filament activation. J Physiol. 1986 Aug;377:487–505. doi: 10.1113/jphysiol.1986.sp016199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moss R. L., Giulian G. G., Greaser M. L. Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers. J Cell Biol. 1983 Apr;96(4):970–978. doi: 10.1083/jcb.96.4.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Moss R. L., Giulian G. G., Greaser M. L. The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers. J Gen Physiol. 1985 Oct;86(4):585–600. doi: 10.1085/jgp.86.4.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Moss R. L., Swinford A. E., Greaser M. L. Alterations in the Ca2+ sensitivity of tension development by single skeletal muscle fibers at stretched lengths. Biophys J. 1983 Jul;43(1):115–119. doi: 10.1016/S0006-3495(83)84329-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mrakovcić A., Oda S., Reisler E. Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin. Biochemistry. 1979 Dec 25;18(26):5960–5965. doi: 10.1021/bi00593a031. [DOI] [PubMed] [Google Scholar]
  34. Oda S., Oriol-Audit C., Reisler E. Effects of actin and calcium ion on chymotryptic digestion of skeletal myosin and their implications to the function of light chains. Biochemistry. 1980 Nov 25;19(24):5614–5618. doi: 10.1021/bi00565a024. [DOI] [PubMed] [Google Scholar]
  35. Pastra-Landis S. C., Lowey S. Myosin subunit interactions. Properties of the 19,000-dalton light chain-deficient myosin. J Biol Chem. 1986 Nov 5;261(31):14811–14816. [PubMed] [Google Scholar]
  36. 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]
  37. Perrie W. T., Smillie L. B., Perry S. B. A phosphorylated light-chain component of myosin from skeletal muscle. Biochem J. 1973 Sep;135(1):151–164. doi: 10.1042/bj1350151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Persechini A., Rowe A. J. Modulation of myosin filament conformation by physiological levels of divalent cation. J Mol Biol. 1984 Jan 5;172(1):23–39. doi: 10.1016/0022-2836(84)90412-1. [DOI] [PubMed] [Google Scholar]
  39. Persechini A., Stull J. T. Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity. Biochemistry. 1984 Aug 28;23(18):4144–4150. doi: 10.1021/bi00313a021. [DOI] [PubMed] [Google Scholar]
  40. Pulliam D. L., Sawyna V., Levine R. J. Calcium sensitivity of vertebrate skeletal muscle myosin. Biochemistry. 1983 May 10;22(10):2324–2331. doi: 10.1021/bi00279a004. [DOI] [PubMed] [Google Scholar]
  41. Reiser P. J., Moss R. L., Giulian G. G., Greaser M. L. Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition. J Biol Chem. 1985 Aug 5;260(16):9077–9080. [PubMed] [Google Scholar]
  42. Robertson S. P., Johnson J. D., Potter J. D. The time-course of Ca2+ exchange with calmodulin, troponin, parvalbumin, and myosin in response to transient increases in Ca2+. Biophys J. 1981 Jun;34(3):559–569. doi: 10.1016/S0006-3495(81)84868-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sweeney H. L., Kushmerick M. J., Mabuchi K., Sréter F. A., Gergely J. Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers. J Biol Chem. 1988 Jun 25;263(18):9034–9039. [PubMed] [Google Scholar]
  44. Sweeney H. L., Stull J. T. Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells. Am J Physiol. 1986 Apr;250(4 Pt 1):C657–C660. doi: 10.1152/ajpcell.1986.250.4.C657. [DOI] [PubMed] [Google Scholar]
  45. Tawada K., Kimura M. Stiffness of glycerinated rabbit psoas fibers in the rigor state. Filament-overlap relation. Biophys J. 1984 Mar;45(3):593–602. doi: 10.1016/S0006-3495(84)84197-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tokunaga M., Suzuki M., Saeki K., Wakabayashi T. Position of the amino terminus of myosin light chain 1 and light chain 2 determined by electron microscopy with monoclonal antibody. J Mol Biol. 1987 Mar 20;194(2):245–255. doi: 10.1016/0022-2836(87)90372-x. [DOI] [PubMed] [Google Scholar]
  47. Ueno H., Harrington W. F. Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge. J Mol Biol. 1986 Jul 5;190(1):59–68. doi: 10.1016/0022-2836(86)90075-6. [DOI] [PubMed] [Google Scholar]
  48. Wagner P. D. Effect of skeletal muscle myosin light chain 2 on the Ca2+-sensitive interaction of myosin and heavy meromyosin with regulated actin. Biochemistry. 1984 Dec 4;23(25):5950–5956. doi: 10.1021/bi00320a010. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Wagner P. D. Preparation and fractionation of myosin light chains and exchange of the essential light chains. Methods Enzymol. 1982;85(Pt B):72–81. doi: 10.1016/0076-6879(82)85010-6. [DOI] [PubMed] [Google Scholar]
  51. Waller G. S., Lowey S. Myosin subunit interactions. Localization of the alkali light chains. J Biol Chem. 1985 Nov 15;260(26):14368–14373. [PubMed] [Google Scholar]
  52. Weeds A. G. Light chains of myosin. Nature. 1969 Sep 27;223(5213):1362–1364. doi: 10.1038/2231362a0. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Winkelmann D. A., Lowey S., Press J. L. Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell. 1983 Aug;34(1):295–306. doi: 10.1016/0092-8674(83)90160-5. [DOI] [PubMed] [Google Scholar]
  55. Winkelmann D. A., Lowey S. Probing myosin head structure with monoclonal antibodies. J Mol Biol. 1986 Apr 20;188(4):595–612. doi: 10.1016/s0022-2836(86)80009-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES