Purification of Native Myosin Filaments from Muscle

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2001 Oct 19

PMID 11606293

Citations 14

Authors

C Hidalgo

R Padron

R Horowitz

F Q Zhao

R Craig

Affiliations

Soon will be listed here.

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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References

Padron R, Granados M, Alamo L, Guerrero J, Craig R . Visualization of myosin helices in sections of rapidly frozen relaxed tarantula muscle. J Struct Biol. 1992; 108(3):269-76. DOI: 10.1016/1047-8477(92)90027-8. View

Selden L, KINOSIAN H, Newman J, LINCOLN B, HURWITZ C, Gershman L . Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin. Biophys J. 1998; 75(6):3092-100. PMC: 1299980. DOI: 10.1016/S0006-3495(98)77750-1. View

Sweeney H, Bowman B, Stull J . Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol. 1993; 264(5 Pt 1):C1085-95. DOI: 10.1152/ajpcell.1993.264.5.C1085. View

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; 65(2):799-805. PMC: 1225780. DOI: 10.1016/S0006-3495(93)81121-4. View

Allen P, Janmey P . 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; 269(52):32916-23. View

Padron R, Alamo L, Guerrero J, Granados M, Uman P, Craig R . Three-dimensional reconstruction of thick filaments from rapidly frozen, freeze-substituted tarantula muscle. J Struct Biol. 1995; 115(3):250-7. DOI: 10.1006/jsbi.1995.1049. View

Dabrowska R, Hinssen H, Galazkiewicz 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; 315 ( Pt 3):753-9. PMC: 1217271. DOI: 10.1042/bj3150753. View

Xu J, Harder B, Uman P, Craig R . Myosin filament structure in vertebrate smooth muscle. J Cell Biol. 1996; 134(1):53-66. PMC: 2120914. DOI: 10.1083/jcb.134.1.53. View

Owen C, Morgan D, DeRosier D . Image analysis of helical objects: the Brandeis Helical Package. J Struct Biol. 1996; 116(1):167-75. DOI: 10.1006/jsbi.1996.0027. View

10.

LEVINE R, Kensler R, Yang Z, Stull J, Sweeney H . Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments. Biophys J. 1996; 71(2):898-907. PMC: 1233547. DOI: 10.1016/S0006-3495(96)79293-7. View

11.

Xu S, Malinchik S, Gilroy D, Kraft T, Brenner B, Yu L . X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys J. 1997; 73(5):2292-303. PMC: 1181134. DOI: 10.1016/S0006-3495(97)78261-4. View

12.

Pope B, Gooch J, Weeds A . Probing the effects of calcium on gelsolin. Biochemistry. 1998; 36(50):15848-55. DOI: 10.1021/bi972192p. View

13.

Padron R, Alamo L, Murgich J, Craig R . Towards an atomic model of the thick filaments of muscle. J Mol Biol. 1998; 275(1):35-41. DOI: 10.1006/jmbi.1997.1448. View

14.

Offer G, Knight P, Burgess S, Alamo L, Padron R . A new model for the surface arrangement of myosin molecules in tarantula thick filaments. J Mol Biol. 2000; 298(2):239-60. DOI: 10.1006/jmbi.2000.3664. View

15.

Maciver S, Ternent D, McLaughlin P . Domain 2 of gelsolin binds directly to tropomyosin. FEBS Lett. 2000; 473(1):71-5. DOI: 10.1016/s0014-5793(00)01507-6. View

16.

Hidalgo C, Craig R, Ikebe M, Padron R . Mechanism of phosphorylation of the regulatory light chain of myosin from tarantula striated muscle. J Muscle Res Cell Motil. 2001; 22(1):51-9. DOI: 10.1023/a:1010388103354. View

17.

Ohta Y . Thermostable protease from thermophilic bacteria. II. Studies on the stability of the protease. J Biol Chem. 1967; 242(3):509-15. View

18.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

19.

Hardwicke P, Hanson J . Separation of thick and thin myofilaments. J Mol Biol. 1971; 59(3):509-16. DOI: 10.1016/0022-2836(71)90314-7. View

20.

Morimoto K, Harrington W . Isolation and composition of thick filaments from rabbit skeletal muscle. J Mol Biol. 1973; 77(1):165-75. DOI: 10.1016/0022-2836(73)90370-7. View