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. 1991 May 1;113(3):563–572. doi: 10.1083/jcb.113.3.563

Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments

PMCID: PMC2288973  PMID: 2016336

Abstract

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick- Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)- independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end- fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.

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Selected References

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  1. Adelstein R. S., Klee C. B. Purification of smooth muscle myosin light-chain kinase. Methods Enzymol. 1982;85(Pt B):298–308. doi: 10.1016/0076-6879(82)85029-5. [DOI] [PubMed] [Google Scholar]
  2. Bennett A. J., Patel N., Wells C., Bagshaw C. R. 8-Anilino-1-naphthalenesulphonate, a fluorescent probe for the regulatory light chain binding site of scallop myosin. J Muscle Res Cell Motil. 1984 Apr;5(2):165–182. doi: 10.1007/BF00712154. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Craig R., Padrón R., Kendrick-Jones J. Structural changes accompanying phosphorylation of tarantula muscle myosin filaments. J Cell Biol. 1987 Sep;105(3):1319–1327. doi: 10.1083/jcb.105.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dewey M. M., Levine R. J., Colflesh D. E. Structure of Limulus striated muscle. The contractile apparatus at various sarcomere lengths. J Cell Biol. 1973 Sep;58(3):574–593. doi: 10.1083/jcb.58.3.574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Epstein H. F., Ortiz I., Mackinnon L. A. The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans. J Cell Biol. 1986 Sep;103(3):985–993. doi: 10.1083/jcb.103.3.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frado L. L., Craig R. Structural changes induced in Ca2+-regulated myosin filaments by Ca2+ and ATP. J Cell Biol. 1989 Aug;109(2):529–538. doi: 10.1083/jcb.109.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hoyle G., McAlear J. H., Selverston A. Mechanism of supercontraction in a striated muscle. J Cell Biol. 1965 Aug;26(2):621–640. doi: 10.1083/jcb.26.2.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kensler R. W., Levine R. J. An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments. J Cell Biol. 1982 Feb;92(2):443–451. doi: 10.1083/jcb.92.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kerrick W. G., Bolles L. L. Regulation of Ca2+-activated tension in limulus striated muscle. Pflugers Arch. 1981 Dec;392(2):121–124. doi: 10.1007/BF00581259. [DOI] [PubMed] [Google Scholar]
  12. Lehman W., Szent-Györgyi A. G. Regulation of muscular contraction. Distribution of actin control and myosin control in the animal kingdom. J Gen Physiol. 1975 Jul;66(1):1–30. doi: 10.1085/jgp.66.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Levine R. J., Chantler P. D., Kensler R. W., Woodhead J. L. Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments. J Cell Biol. 1991 May;113(3):563–572. doi: 10.1083/jcb.113.3.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Levine R. J., Davidheiser S., Kelly A. M., Kensler R. W., Leferovich J., Davies R. E. Fibre types in Limulus telson muscles: morphology and histochemistry. J Muscle Res Cell Motil. 1989 Feb;10(1):53–66. doi: 10.1007/BF01739856. [DOI] [PubMed] [Google Scholar]
  15. Levine R. J., Kensler R. W., Levitt P. Crossbridge and backbone structure of invertebrate thick filaments. Biophys J. 1986 Jan;49(1):135–138. doi: 10.1016/S0006-3495(86)83624-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Levine R. J., Kensler R. W. Structure of short thick filaments from Limulus muscle. J Mol Biol. 1985 Mar 20;182(2):347–352. doi: 10.1016/0022-2836(85)90351-1. [DOI] [PubMed] [Google Scholar]
  17. Leyton R. A., Ullrick W. G. Z disc ultrastructure in scutal depressor fibers of the barnacle. Science. 1970 Apr 3;168(3927):127–128. doi: 10.1126/science.168.3927.127. [DOI] [PubMed] [Google Scholar]
  18. Miller D. M., 3rd, Ortiz I., Berliner G. C., Epstein H. F. Differential localization of two myosins within nematode thick filaments. Cell. 1983 Sep;34(2):477–490. doi: 10.1016/0092-8674(83)90381-1. [DOI] [PubMed] [Google Scholar]
  19. Perkins F. O., Ramsey R. W., Street S. F. The ultrastructure of fishing tentacle muscle in the jellyfish Chrysaora quinquecirrha: a comparison of contracted and relaxed states. J Ultrastruct Res. 1971 Jun;35(5):431–450. doi: 10.1016/s0022-5320(71)80004-7. [DOI] [PubMed] [Google Scholar]
  20. Sellers J. R. Phosphorylation-dependent regulation of Limulus myosin. J Biol Chem. 1981 Sep 10;256(17):9274–9278. [PubMed] [Google Scholar]
  21. Szentkiralyi E. M. Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide. J Muscle Res Cell Motil. 1984 Apr;5(2):147–164. doi: 10.1007/BF00712153. [DOI] [PubMed] [Google Scholar]
  22. Vibert P., Craig R. Structural changes that occur in scallop myosin filaments upon activation. J Cell Biol. 1985 Sep;101(3):830–837. doi: 10.1083/jcb.101.3.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Walsh M. P., Dabrowska R., Hinkins S., Hartshorne D. J. Calcium-independent myosin light chain kinase of smooth muscle. Preparation by limited chymotryptic digestion of the calcium ion dependent enzyme, purification, and characterization. Biochemistry. 1982 Apr 13;21(8):1919–1925. doi: 10.1021/bi00537a034. [DOI] [PubMed] [Google Scholar]
  24. Winkelmann D. A., Almeda S., Vibert P., Cohen C. A new myosin fragment: visualization of the regulatory domain. Nature. 1984 Feb 23;307(5953):758–760. doi: 10.1038/307758a0. [DOI] [PubMed] [Google Scholar]
  25. Wray J. S., Vibert P. J., Cohen C. Cross-bridge arrangements in Limulus muscle. J Mol Biol. 1974 Sep 15;88(2):343–348. doi: 10.1016/0022-2836(74)90486-0. [DOI] [PubMed] [Google Scholar]

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