Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1982 Dec 1;95(3):763–770. doi: 10.1083/jcb.95.3.763

Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro

PMCID: PMC2112936  PMID: 6185504

Abstract

Monoclonal antibodies (McAbs) against the myosin heavy chain (MHC) of adult chicken pectoralis muscle have been tested for reactivity with pectoralis myosin at selected stages of chick development in vivo and in vitro. Three such McAbs, MF 20 and MF 14, which bind to light meromyosin, and MF 30, which binds to myosin subfragment two (S2), were used to assay the appearance and accumulation of specific MHC epitopes with: (a) indirect, solid phase radioimmune assay (RIA), (b) immunoautoradiography, (c) immunofluorescence microscopy. McAb MF 20 bound strongly and equivalently to MHC at all stages of embryonic development in vivo. In contrast, the MF 30 epitope was barely detectable at 12 d of incubation but its concentration rose rapidly just before hatching. No detectable binding of MF 14 to pectoralis myosin could be measured during myogenesis in vivo until 1 wk after hatching. Immunofluorescence studies revealed that all three epitopes accumulate in the same myocytes of the developing pectoralis muscle. Since all three McAbs bound with high activity to native and denatured forms of myosin, it is unlikely that differential antibody reactivity can be explained by conformational changes in myosin during development in vivo. When myogenesis in vitro was monitored using the same McAbs, MF 20 bound to the MHC at all stages tested while reactivity of MF 30 and MF 14 with myosin from cultured muscle was never observed. Thus, this study demonstrates three different immunochemical states of the MHC during development in vivo of chick pectoralis muscle and the absence of later occurring immunochemical transitions in the MHC of cultured embryonic muscle.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Arndt I., Pepe F. A. Antigenic specificity of red and white muscle myosin. J Histochem Cytochem. 1975 Mar;23(3):159–168. doi: 10.1177/23.3.47867. [DOI] [PubMed] [Google Scholar]
  2. Bandman E., Matsuda R., Micou-Eastwood J., Strohman R. In vitro translation of RNA from embryonic and from adult chicken pectoralis muscle produces different myosin heavy chains. FEBS Lett. 1981 Dec 28;136(2):301–305. doi: 10.1016/0014-5793(81)80640-0. [DOI] [PubMed] [Google Scholar]
  3. Bruggmann S., Jenny E. The immunological specificity of myosins from cross-striated muscles as revealed by quantitative microcomplement fixation and enzyme inhibition by antisera. Biochim Biophys Acta. 1975 Nov 18;412(1):39–50. doi: 10.1016/0005-2795(75)90337-2. [DOI] [PubMed] [Google Scholar]
  4. Bullaro J. C., Brookman D. H. Comparison of skeletal muscle monolayer cultures initiated with cells dissociated by the vortex and trypsin methods. In Vitro. 1976 Aug;12(8):564–570. doi: 10.1007/BF02797440. [DOI] [PubMed] [Google Scholar]
  5. Devlin R. B., Emerson C. P., Jr Coordinate accumulation of contractile protein mRNAs during myoblast differentiation. Dev Biol. 1979 Mar;69(1):202–216. doi: 10.1016/0012-1606(79)90286-0. [DOI] [PubMed] [Google Scholar]
  6. Devlin R. B., Emerson C. P., Jr Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell. 1978 Apr;13(4):599–611. doi: 10.1016/0092-8674(78)90211-8. [DOI] [PubMed] [Google Scholar]
  7. Dhoot G. K., Perry S. V. Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle. Nature. 1979 Apr 19;278(5706):714–718. doi: 10.1038/278714a0. [DOI] [PubMed] [Google Scholar]
  8. Fenner C., Traut R. R., Mason D. T., Wikman-Coffelt J. Quantification of Coomassie Blue stained proteins in polyacrylamide gels based on analyses of eluted dye. Anal Biochem. 1975 Feb;63(2):595–602. doi: 10.1016/0003-2697(75)90386-3. [DOI] [PubMed] [Google Scholar]
  9. Galfre G., Howe S. C., Milstein C., Butcher G. W., Howard J. C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature. 1977 Apr 7;266(5602):550–552. doi: 10.1038/266550a0. [DOI] [PubMed] [Google Scholar]
  10. Gauthier G. F., Lowey S., Benfield P. A., Hobbs A. W. Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers. J Cell Biol. 1982 Feb;92(2):471–484. doi: 10.1083/jcb.92.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gauthier G. F., Lowey S. Distribution of myosin isoenzymes among skeletal muscle fiber types. J Cell Biol. 1979 Apr;81(1):10–25. doi: 10.1083/jcb.81.1.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gauthier G. F., Lowey S., Hobbs A. W. Fast and slow myosin in developing muscle fibres. Nature. 1978 Jul 6;274(5666):25–29. doi: 10.1038/274025a0. [DOI] [PubMed] [Google Scholar]
  13. Gefter M. L., Margulies D. H., Scharff M. D. A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells. Somatic Cell Genet. 1977 Mar;3(2):231–236. doi: 10.1007/BF01551818. [DOI] [PubMed] [Google Scholar]
  14. Hoh J. F., Yeoh G. P. Rabbit skeletal myosin isoenzymes from fetal, fast-twitch and slow-twitch muscles. Nature. 1979 Jul 26;280(5720):321–323. doi: 10.1038/280321a0. [DOI] [PubMed] [Google Scholar]
  15. Huszar G. Developmental changes of the primary structure and histidine methylation in rabbit skeletal muscle myosin. Nat New Biol. 1972 Dec 27;240(104):260–264. doi: 10.1038/newbio240260a0. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. 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]
  18. Lompre A. M., Bouveret P., Leger J., Schwartz K. Detection of antibodies specific to sodium dodecyl sulfate-treated proteins. J Immunol Methods. 1979;28(1-2):143–148. doi: 10.1016/0022-1759(79)90336-3. [DOI] [PubMed] [Google Scholar]
  19. Lutz H., Weber H., Billeter R., Jenny E. Fast and slow myosin within single skeletal muscle fibres of adult rabbits. Nature. 1979 Sep 13;281(5727):142–144. doi: 10.1038/281142a0. [DOI] [PubMed] [Google Scholar]
  20. Masaki T., Yoshizaki C. Differentiation of myosin in chick embryos. J Biochem. 1974 Jul;76(1):123–131. doi: 10.1093/oxfordjournals.jbchem.a130536. [DOI] [PubMed] [Google Scholar]
  21. Matsuda R., Obinata T., Shimada Y. Types of troponin components during development of chicken skeletal muscle. Dev Biol. 1981 Feb;82(1):11–19. doi: 10.1016/0012-1606(81)90424-3. [DOI] [PubMed] [Google Scholar]
  22. Morris R. J., Williams A. F. Antigens on mouse and rat lymphocytes recognized by rabbit antiserum against rat brain: the quantitative analysis of a xenogeneic antiserum. Eur J Immunol. 1975 Apr;5(4):274–281. doi: 10.1002/eji.1830050412. [DOI] [PubMed] [Google Scholar]
  23. Pope B. J., Wagner P. D., Weeds A. G. Heterogeneity of myosin heavy chains in subfragment-1 isoenzymes rabbit skeletal myosin. J Mol Biol. 1977 Jan 25;109(3):470–473. doi: 10.1016/s0022-2836(77)80024-7. [DOI] [PubMed] [Google Scholar]
  24. Reinach F. C., Masaki T., Shafiq S., Obinata T., Fischman D. A. Isoforms of C-protein in adult chicken skeletal muscle: detection with monoclonal antibodies. J Cell Biol. 1982 Oct;95(1):78–84. doi: 10.1083/jcb.95.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roy R. K., Sreter F. A., Sarkar S. Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit striated muscles. Dev Biol. 1979 Mar;69(1):15–30. doi: 10.1016/0012-1606(79)90271-9. [DOI] [PubMed] [Google Scholar]
  26. Rubinstein N. A., Kelly A. M. Development of muscle fiber specialization in the rat hindlimb. J Cell Biol. 1981 Jul;90(1):128–144. doi: 10.1083/jcb.90.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schwartz K., Bouveret P., Sebag C., Leger J., Swynghedauw B. Immunochemical evidence for the species-specificity of mammalian cardiac myosin and heavy meromyosin. Biochim Biophys Acta. 1977 Nov 25;495(1):24–36. doi: 10.1016/0005-2795(77)90236-7. [DOI] [PubMed] [Google Scholar]
  28. Shimada Y., Fischman D. A., Moscona A. A. The fine structure of embryonic chick skeletal muscle cells differentiated in vitro. J Cell Biol. 1967 Nov;35(2):445–453. doi: 10.1083/jcb.35.2.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sréter F. A., Bálint M., Gergely J. Structural and functional changes of myosin during development: comparison with adult fast, slow and cardiac myosin. Dev Biol. 1975 Oct;46(2):317–325. doi: 10.1016/0012-1606(75)90108-6. [DOI] [PubMed] [Google Scholar]
  30. Starr R., Offer G. Polarity of the myosin molecule. J Mol Biol. 1973 Nov 25;81(1):17–31. doi: 10.1016/0022-2836(73)90244-1. [DOI] [PubMed] [Google Scholar]
  31. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Whalen R. G., Schwartz K., Bouveret P., Sell S. M., Gros F. Contractile protein isozymes in muscle development: identification of an embryonic form of myosin heavy chain. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5197–5201. doi: 10.1073/pnas.76.10.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Whalen R. G., Sell S. M., Butler-Browne G. S., Schwartz K., Bouveret P., Pinset-Härstöm I. Three myosin heavy-chain isozymes appear sequentially in rat muscle development. Nature. 1981 Aug 27;292(5826):805–809. doi: 10.1038/292805a0. [DOI] [PubMed] [Google Scholar]
  34. Wikman-Coffelt J., Fenner C., Smith A., Mason D. T. Comparative analyses of the kinetics and subunits of myosins from canine skeletal muscle and cardiac tissue. J Biol Chem. 1975 Feb 25;250(4):1257–1262. [PubMed] [Google Scholar]
  35. Yoshida M., Katoh N., Kubo S., Yagi K. Immunochemically detected structural difference in the heavy chains of chicken breast muscle myosin isozymes. J Biochem. 1981 Apr;89(4):991–997. [PubMed] [Google Scholar]
  36. Zweig S. E. The muscle specificity and structure of two closely related fast-twitch white muscle myosin heavy chain isozymes. J Biol Chem. 1981 Nov 25;256(22):11847–11853. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES