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. 2001 Nov;81(5):2817–2826. doi: 10.1016/S0006-3495(01)75923-1

Purification of native myosin filaments from muscle.

C Hidalgo 1, R Padrón 1, R Horowitz 1, F Q Zhao 1, R Craig 1
PMCID: PMC1301747  PMID: 11606293

Abstract

Analysis of the structure and function of native thick (myosin-containing) filaments of muscle has been hampered in the past by the difficulty of obtaining a pure preparation. We have developed a simple method for purifying native myosin filaments from muscle filament suspensions. The method involves severing thin (actin-containing) filaments into short segments using a Ca(2+)-insensitive fragment of gelsolin, followed by differential centrifugation to purify the thick filaments. By gel electrophoresis, the purified thick filaments show myosin heavy and light chains together with nonmyosin thick filament components. Contamination with actin is below 3.5%. Electron microscopy demonstrates intact thick filaments, with helical cross-bridge order preserved, and essentially complete removal of thin filaments. The method has been developed for striated muscles but can also be used in a modified form to remove contaminating thin filaments from native smooth muscle myofibrils. Such preparations should be useful for thick filament structural and biochemical studies.

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Selected References

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  1. Allen P. G., Janmey P. A. Gelsolin displaces phalloidin from actin filaments. A new fluorescence method shows that both Ca2+ and Mg2+ affect the rate at which gelsolin severs F-actin. J Biol Chem. 1994 Dec 30;269(52):32916–32923. [PubMed] [Google Scholar]
  2. Blanchard A., Ohanian V., Critchley D. The structure and function of alpha-actinin. J Muscle Res Cell Motil. 1989 Aug;10(4):280–289. doi: 10.1007/BF01758424. [DOI] [PubMed] [Google Scholar]
  3. Brenner B., Schoenberg M., Chalovich J. M., Greene L. E., Eisenberg E. Evidence for cross-bridge attachment in relaxed muscle at low ionic strength. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7288–7291. doi: 10.1073/pnas.79.23.7288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brenner B., Yu L. C., Podolsky R. J. X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths. Biophys J. 1984 Sep;46(3):299–306. doi: 10.1016/S0006-3495(84)84026-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bryan J., Hwo S. Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin. J Cell Biol. 1986 Apr;102(4):1439–1446. doi: 10.1083/jcb.102.4.1439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Castellani L., Hardwicke P. M. Crystalline structure of sarcoplasmic reticulum from scallop. J Cell Biol. 1983 Aug;97(2):557–561. doi: 10.1083/jcb.97.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castellani L., Hardwicke P. M., Vibert P. Dimer ribbons in the three-dimensional structure of sarcoplasmic reticulum. J Mol Biol. 1985 Oct 5;185(3):579–594. doi: 10.1016/0022-2836(85)90073-7. [DOI] [PubMed] [Google Scholar]
  8. Chaponnier C., Janmey P. A., Yin H. L. The actin filament-severing domain of plasma gelsolin. J Cell Biol. 1986 Oct;103(4):1473–1481. doi: 10.1083/jcb.103.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooke P. H., Fay F. S., Craig R. Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar. J Muscle Res Cell Motil. 1989 Jun;10(3):206–220. doi: 10.1007/BF01739811. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Crowther R. A., Padrón R., Craig R. Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle. J Mol Biol. 1985 Aug 5;184(3):429–439. doi: 10.1016/0022-2836(85)90292-x. [DOI] [PubMed] [Google Scholar]
  12. Dabrowska R., Hinssen H., Gałazkiewicz B., Nowak E. Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity. Biochem J. 1996 May 1;315(Pt 3):753–759. doi: 10.1042/bj3150753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emes C. H., Rowe A. J. Frictional properties and molecular weight of native and synthetic myosin filaments from vertebrate skeletal muscle. Biochim Biophys Acta. 1978 Nov 20;537(1):125–144. doi: 10.1016/0005-2795(78)90608-6. [DOI] [PubMed] [Google Scholar]
  14. Fattoum A., Hartwig J. H., Stossel T. P. Isolation and some structural and functional properties of macrophage tropomyosin. Biochemistry. 1983 Mar 1;22(5):1187–1193. doi: 10.1021/bi00274a031. [DOI] [PubMed] [Google Scholar]
  15. Gregory D. W., Pirie B. J. Wetting agents for biological electron microscopy. I. General considerations and negative staining. J Microsc. 1973 Dec;99(3):251–255. doi: 10.1111/j.1365-2818.1973.tb04625.x. [DOI] [PubMed] [Google Scholar]
  16. Hardwicke P. M., Hanson J. Separation of thick and thin myofilaments. J Mol Biol. 1971 Aug 14;59(3):509–516. doi: 10.1016/0022-2836(71)90314-7. [DOI] [PubMed] [Google Scholar]
  17. Hellweg T., Hinssen H., Eimer W. The Ca(2+)-induced conformational change of gelsolin is located in the carboxyl-terminal half of the molecule. Biophys J. 1993 Aug;65(2):799–805. doi: 10.1016/S0006-3495(93)81121-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hidalgo C., Craig R., Ikebe M., Padrón R. Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle. J Muscle Res Cell Motil. 2001;22(1):51–59. doi: 10.1023/a:1010388103354. [DOI] [PubMed] [Google Scholar]
  19. Hori M., Karaki H. Regulatory mechanisms of calcium sensitization of contractile elements in smooth muscle. Life Sci. 1998;62(17-18):1629–1633. doi: 10.1016/s0024-3205(98)00119-2. [DOI] [PubMed] [Google Scholar]
  20. Ishikawa R., Yamashiro S., Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989 May 5;264(13):7490–7497. [PubMed] [Google Scholar]
  21. Janmey P. A., Iida K., Yin H. L., Stossel T. P. Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 1987 Sep 5;262(25):12228–12236. [PubMed] [Google Scholar]
  22. 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]
  23. Kurokawa H., Fujii W., Ohmi K., Sakurai T., Nonomura Y. Simple and rapid purification of brevin. Biochem Biophys Res Commun. 1990 Apr 30;168(2):451–457. doi: 10.1016/0006-291x(90)92342-w. [DOI] [PubMed] [Google Scholar]
  24. Kwiatkowski D. J., Janmey P. A., Mole J. E., Yin H. L. Isolation and properties of two actin-binding domains in gelsolin. J Biol Chem. 1985 Dec 5;260(28):15232–15238. [PubMed] [Google Scholar]
  25. Kwiatkowski D. J., Stossel T. P., Orkin S. H., Mole J. E., Colten H. R., Yin H. L. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 1986 Oct 2;323(6087):455–458. doi: 10.1038/323455a0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lamb J. A., Allen P. G., Tuan B. Y., Janmey P. A. Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement. J Biol Chem. 1993 Apr 25;268(12):8999–9004. [PubMed] [Google Scholar]
  28. Langer M., Giebing T., D'Haese J. Purification and functional characterization of an 85-kDa gelsolin from the ascidians Microcosmus sulcatus and Phallusia mammilata. Comp Biochem Physiol B Biochem Mol Biol. 1998 Apr;119(4):697–704. doi: 10.1016/s0305-0491(98)00045-5. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Levine R. J., Kensler R. W., Reedy M. C., Hofmann W., King H. A. Structure and paramyosin content of tarantula thick filaments. J Cell Biol. 1983 Jul;97(1):186–195. doi: 10.1083/jcb.97.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Levine R. J., Kensler R. W., Yang Z., Stull J. T., Sweeney H. L. Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments. Biophys J. 1996 Aug;71(2):898–907. doi: 10.1016/S0006-3495(96)79293-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Maciver S. K., Ternent D., McLaughlin P. J. Domain 2 of gelsolin binds directly to tropomyosin. FEBS Lett. 2000 May 4;473(1):71–75. doi: 10.1016/s0014-5793(00)01507-6. [DOI] [PubMed] [Google Scholar]
  33. Morimoto K., Harrington W. F. Isolation and composition of thick filaments from rabbit skeletal muscle. J Mol Biol. 1973 Jun 15;77(1):165–175. doi: 10.1016/0022-2836(73)90370-7. [DOI] [PubMed] [Google Scholar]
  34. Offer G., Knight P. J., Burgess S. A., Alamo L., Padrón R. A new model for the surface arrangement of myosin molecules in tarantula thick filaments. J Mol Biol. 2000 Apr 28;298(2):239–260. doi: 10.1006/jmbi.2000.3664. [DOI] [PubMed] [Google Scholar]
  35. Ohta Y. Thermostable protease from thermophilic bacteria. II. Studies on the stability of the protease. J Biol Chem. 1967 Feb 10;242(3):509–515. [PubMed] [Google Scholar]
  36. Owen C. H., Morgan D. G., DeRosier D. J. Image analysis of helical objects: the Brandeis Helical Package. J Struct Biol. 1996 Jan-Feb;116(1):167–175. doi: 10.1006/jsbi.1996.0027. [DOI] [PubMed] [Google Scholar]
  37. Padrón R., Alamo L., Guerrero J. R., Granados M., Uman P., Craig R. Three-dimensional reconstruction of thick filaments from rapidly frozen, freeze-substituted tarantula muscle. J Struct Biol. 1995 Nov-Dec;115(3):250–257. doi: 10.1006/jsbi.1995.1049. [DOI] [PubMed] [Google Scholar]
  38. Padrón R., Alamo L., Murgich J., Craig R. Towards an atomic model of the thick filaments of muscle. J Mol Biol. 1998 Jan 9;275(1):35–41. doi: 10.1006/jmbi.1997.1448. [DOI] [PubMed] [Google Scholar]
  39. Padrón R., Granados M., Alamo L., Guerrero J. R., Craig R. Visualization of myosin helices in sections of rapidly frozen relaxed tarantula muscle. J Struct Biol. 1992 May-Jun;108(3):269–276. doi: 10.1016/1047-8477(92)90027-8. [DOI] [PubMed] [Google Scholar]
  40. Pope B. J., Gooch J. T., Weeds A. G. Probing the effects of calcium on gelsolin. Biochemistry. 1997 Dec 16;36(50):15848–15855. doi: 10.1021/bi972192p. [DOI] [PubMed] [Google Scholar]
  41. Scales D., Giuseppeinesi Assembly of ATPase protein in sarcoplasmic reticulum membranes. Biophys J. 1976 Jul;16(7):735–751. doi: 10.1016/S0006-3495(76)85725-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schoepper B., Wegner A. Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin-actin complex. Eur J Biochem. 1991 Dec 18;202(3):1127–1131. doi: 10.1111/j.1432-1033.1991.tb16480.x. [DOI] [PubMed] [Google Scholar]
  43. Selden L. A., Kinosian H. J., Newman J., Lincoln B., Hurwitz C., Gershman L. C., Estes J. E. Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin. Biophys J. 1998 Dec;75(6):3092–3100. doi: 10.1016/S0006-3495(98)77750-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sellers J. R. Phosphorylation-dependent regulation of Limulus myosin. J Biol Chem. 1981 Sep 10;256(17):9274–9278. [PubMed] [Google Scholar]
  45. Soua Z., Porte F., Harricane M. C., Feinberg J., Capony J. P. Bovine serum brevin. Purification by hydrophobic chromatography and properties. Eur J Biochem. 1985 Dec 2;153(2):275–287. doi: 10.1111/j.1432-1033.1985.tb09298.x. [DOI] [PubMed] [Google Scholar]
  46. Suda H., Aoyagi T., Takeuchi T., Umezawa H. Letter: A thermolysin inhibitor produced by Actinomycetes: phospholamidon. J Antibiot (Tokyo) 1973 Oct;26(10):621–623. doi: 10.7164/antibiotics.26.621. [DOI] [PubMed] [Google Scholar]
  47. Sutoh K., Yin H. L. End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin. Biochemistry. 1989 Jun 13;28(12):5269–5275. doi: 10.1021/bi00438a052. [DOI] [PubMed] [Google Scholar]
  48. Suzuki H., Onishi H., Takahashi K., Watanabe S. Structure and function of chicken gizzard myosin. J Biochem. 1978 Dec;84(6):1529–1542. doi: 10.1093/oxfordjournals.jbchem.a132278. [DOI] [PubMed] [Google Scholar]
  49. Sweeney H. L., Bowman B. F., Stull J. T. Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol. 1993 May;264(5 Pt 1):C1085–C1095. doi: 10.1152/ajpcell.1993.264.5.C1085. [DOI] [PubMed] [Google Scholar]
  50. Trinick J. A. Preparation of native thick filaments. Methods Enzymol. 1982;85(Pt B):17–20. doi: 10.1016/0076-6879(82)85007-6. [DOI] [PubMed] [Google Scholar]
  51. Vibert P., Craig R. Electron microscopy and image analysis of myosin filaments from scallop striated muscle. J Mol Biol. 1983 Apr 5;165(2):303–320. doi: 10.1016/s0022-2836(83)80259-9. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Vibert P. Helical reconstruction of frozen-hydrated scallop myosin filaments. J Mol Biol. 1992 Feb 5;223(3):661–671. doi: 10.1016/0022-2836(92)90982-p. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Xu J. Q., Harder B. A., Uman P., Craig R. Myosin filament structure in vertebrate smooth muscle. J Cell Biol. 1996 Jul;134(1):53–66. doi: 10.1083/jcb.134.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Xu S., Malinchik S., Gilroy D., Kraft T., Brenner B., Yu L. C. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys J. 1997 Nov;73(5):2292–2303. doi: 10.1016/S0006-3495(97)78261-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yin H. L., Stossel T. P. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 1979 Oct 18;281(5732):583–586. doi: 10.1038/281583a0. [DOI] [PubMed] [Google Scholar]
  58. le Maire M., Jorgensen K. E., Roigaard-Petersen H., Moller J. V. Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase. Biochemistry. 1976 Dec 28;15(26):5805–5812. doi: 10.1021/bi00671a018. [DOI] [PubMed] [Google Scholar]

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