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. 1983 Jun 1;96(6):1772–1779. doi: 10.1083/jcb.96.6.1772

Novel staining pattern of skeletal muscle M-lines upon incubation with antibodies against MM-creatine kinase

PMCID: PMC2112439  PMID: 6189843

Abstract

Incubation of chicken skeletal muscle fibers with an excess of anti-M- creatine kinase (CK) immunoglobulin G and an excess of anti-M-CK Fab fragments leads to heavy decoration of the M-line (Wallimann, T., D.C. Turner, and H.M. Eppenberger, 1977, J. Cell Biol. 75:297-317) and to removal of the electron-dense M-line structure (Walliman, T., G. W. Pelloni, D.C. Turner, and H.M. Eppenberger, 1978, Proc. Natl. Acad. Sci. USA., 75:4296-4300), respectively. On the other hand, incubation with low concentrations of monovalent anti-M-CK Fab did not extract but rather decorated the M-line, giving rise to a distinct two-line staining pattern. A similar double-line staining pattern, although less pronounced, was also observed within the M-line of paraformaldehyde- prefixed myogenic cells, which after permeabilization were incubated with low concentrations of divalent anti-M-CK antibody. In both cases, the two decorated lines appearing in the middle of the A-band were spaced axially 42-44 nm apart and correspond most likely to the two M4 and M4' m-bridge rows described by Sjostrom and Squire (1977, J. Mol. Biol., 109:49-68; 1977, J. Microscopy., 111:239-278). It is concluded that the muscle-specific form of creatine kinase, MM-CK, contributes mainly to the electron density of these M4 and M4' m-bridges within the M-line structure. This specific labeling pattern is a further demonstration that CK is an integral part of the M-line.

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Selected References

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  1. Baldwin K. M., Cooke D. A., Cheadle W. G. Enzyme alterations in neonatal heart muscle during development. J Mol Cell Cardiol. 1977 Aug;9(8):651–660. doi: 10.1016/s0022-2828(77)80360-x. [DOI] [PubMed] [Google Scholar]
  2. Botts J., Stone D. B., Wang A. T., Mendelson R. A. Electron paramagnetic resonance and nanosecond fluorescence depolarization studies on creatine-phosphokinase interaction with myosin and its fragments. J Supramol Struct. 1975;3(2):141–145. doi: 10.1002/jss.400030206. [DOI] [PubMed] [Google Scholar]
  3. Caravatti M., Perriard J. C., Eppenberger H. M. Developmental regulation of creatine kinase isoenzymes in myogenic cell cultures from chicken. Biosynthesis of creatine kinase subunits M and B. J Biol Chem. 1979 Feb 25;254(4):1388–1394. [PubMed] [Google Scholar]
  4. Carlsson E., Kjörell U., Thornell L. E., Lambertsson A., Strehler E. Differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart. Eur J Cell Biol. 1982 Apr;27(1):62–73. [PubMed] [Google Scholar]
  5. Chowrashi P. K., Pepe F. A. The Z-band: 85,000-dalton amorphin and alpha-actinin and their relation to structure. J Cell Biol. 1982 Sep;94(3):565–573. doi: 10.1083/jcb.94.3.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eppenberger H. M., Perriard J. C., Rosenberg U. B., Strehler E. E. The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells. J Cell Biol. 1981 May;89(2):185–193. doi: 10.1083/jcb.89.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Etlinger J. D., Zak R., Fischman D. A. Compositional studies of myofibrils from rabbit striated muscle. J Cell Biol. 1976 Jan;68(1):123–141. doi: 10.1083/jcb.68.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FRANZINI-ARMSTRONG C., PORTER K. R. SARCOLEMMAL INVAGINATIONS CONSTITUTING THE T SYSTEM IN FISH MUSCLE FIBERS. J Cell Biol. 1964 Sep;22:675–696. doi: 10.1083/jcb.22.3.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Herasymowych O. S., Mani R. S., Kay C. M., Bradley R. D., Scraba D. G. Ultrastructure studies on the binding of creatine kinase and the 165,000 molecular weight component to the M-band of muscle. J Mol Biol. 1980 Jan 15;136(2):193–198. doi: 10.1016/0022-2836(80)90313-7. [DOI] [PubMed] [Google Scholar]
  10. Holt J. C., Lowey S. An immunological approach to the role of the low molecular weight subunits in myosin. II. Interaction of myosin and its subfragments with antibodies to the light chains. Biochemistry. 1975 Oct 21;14(21):4609–4620. doi: 10.1021/bi00692a008. [DOI] [PubMed] [Google Scholar]
  11. Hopkins S. F., Jr, McCutcheon E. P., Wekstein D. R. Postnatal changes in rat ventricular function. Circ Res. 1973 Jun;32(6):685–691. doi: 10.1161/01.res.32.6.685. [DOI] [PubMed] [Google Scholar]
  12. Houk T. W., Jr, Putman S. V. Location of the creatine phosphokinase binding site of myosin. Biochem Biophys Res Commun. 1973 Dec 19;55(4):1271–1277. doi: 10.1016/s0006-291x(73)80031-2. [DOI] [PubMed] [Google Scholar]
  13. Knappeis G. G., Carlsen F. The ultrastructure of the M line in skeletal muscle. J Cell Biol. 1968 Jul;38(1):202–211. doi: 10.1083/jcb.38.1.202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Landon M. F., Oriol C. Native conformation of M-protein. Biochem Biophys Res Commun. 1975 Jan 20;62(2):241–245. doi: 10.1016/s0006-291x(75)80129-x. [DOI] [PubMed] [Google Scholar]
  15. Luther P., Squire J. Three-dimensional structure of the vertebrate muscle M-region. J Mol Biol. 1978 Nov 5;125(3):313–324. doi: 10.1016/0022-2836(78)90405-9. [DOI] [PubMed] [Google Scholar]
  16. Mani R. S., Kay C. M. Interaction studies of the 165 000 dalton protein component of the M-line with the S2 subfragment of myosin. Biochim Biophys Acta. 1978 Sep 26;536(1):134–141. doi: 10.1016/0005-2795(78)90059-4. [DOI] [PubMed] [Google Scholar]
  17. Mani R. S., Kay C. M. Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase. Biochim Biophys Acta. 1978 Mar 28;533(1):248–256. doi: 10.1016/0005-2795(78)90568-8. [DOI] [PubMed] [Google Scholar]
  18. Mani R. S., Kay C. M. Physicochemical studies on the creatine kinase M-line protein and its interaction with myosin and myosin fragments. Biochim Biophys Acta. 1976 Dec 22;453(2):391–399. doi: 10.1016/0005-2795(76)90134-3. [DOI] [PubMed] [Google Scholar]
  19. Masaki T., Takaiti O., Ebashi S. "M-substance", a new protein constituting the M-line of myofibrils. J Biochem. 1968 Dec;64(6):909–910. doi: 10.1093/oxfordjournals.jbchem.a128975. [DOI] [PubMed] [Google Scholar]
  20. Masaki T., Takaiti O. M-protein. J Biochem. 1974 Feb;75(2):367–380. doi: 10.1093/oxfordjournals.jbchem.a130403. [DOI] [PubMed] [Google Scholar]
  21. Morimoto K., Harrington W. F. Isolation and physical chemical properties of an M-line protein from skeletal muscle. J Biol Chem. 1972 May 25;247(10):3052–3061. [PubMed] [Google Scholar]
  22. PORTER R. R. The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem J. 1959 Sep;73:119–126. doi: 10.1042/bj0730119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Palmer E. G. Antibody localization studies of the M-line in striated muscle. Can J Zool. 1975 Jun;53(6):788–799. doi: 10.1139/z75-093. [DOI] [PubMed] [Google Scholar]
  24. Perriard J. C., Perriard E. R., Eppenberger H. M. Detection and relative quantitation of mRNA for creatine kinase isoenzymes in mRNA from myogenic cell cultures and embryonic chicken tissues. J Biol Chem. 1978 Sep 25;253(18):6529–6535. [PubMed] [Google Scholar]
  25. Sjöström M., Squire J. M. Cryo-ultramicrotomy and myofibrillar fine structure: a review. J Microsc. 1977 Dec;111(3):239–278. doi: 10.1111/j.1365-2818.1977.tb00067.x. [DOI] [PubMed] [Google Scholar]
  26. Sjöström M., Squire J. M. Fine structure of the A-band in cryo-sections. The structure of the A-band of human skeletal muscle fibres from ultra-thin cryo-sections negatively stained. J Mol Biol. 1977 Jan 5;109(1):49–68. doi: 10.1016/s0022-2836(77)80045-4. [DOI] [PubMed] [Google Scholar]
  27. Sommer J. R., Johnson E. A. Cardiac muscle. A comparative ultrastructural study with special reference to frog and chicken hearts. Z Zellforsch Mikrosk Anat. 1969;98(3):437–468. [PubMed] [Google Scholar]
  28. Strehler E. E., Pelloni G., Heizmann C. W., Eppenberger H. M. Biochemical and ultrastructural aspects of Mr 165,000 M-protein in cross-striated chicken muscle. J Cell Biol. 1980 Sep;86(3):775–783. doi: 10.1083/jcb.86.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Turner D. C., Gmür R., Siegrist M., Burckhardt E., Eppenberger H. M. Differentiation in cultures derived from embryonic chicken muscle. I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures. Dev Biol. 1976 Feb;48(2):258–283. doi: 10.1016/0012-1606(76)90090-7. [DOI] [PubMed] [Google Scholar]
  30. Turner D. C., Wallimann T., Eppenberger H. M. A protein that binds specifically to the M-line of skeletal muscle is identified as the muscle form of creatine kinase. Proc Natl Acad Sci U S A. 1973 Mar;70(3):702–705. doi: 10.1073/pnas.70.3.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wallimann T., Kuhn H. J., Pelloni G., Turner D. C., Eppenberger H. M. Localization of creatine kinase isoenzymes in myofibrils. II. Chicken heart muscle. J Cell Biol. 1977 Nov;75(2 Pt 1):318–325. doi: 10.1083/jcb.75.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wallimann T., Pelloni G., Turner D. C., Eppenberger H. M. Monovalent antibodies against MM-creatine kinase remove the M line from myofibrils. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4296–4300. doi: 10.1073/pnas.75.9.4296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wallimann T., Szent-Györgyi A. G. An immunological approach to myosin light-chain function in thick filament linked regulation. 1. Characterization, specificity, and cross-reactivity of anti-scallop myosin heavy- and light-chain antibodies by competitive, solid-phase radioimmunoassay. Biochemistry. 1981 Mar 3;20(5):1176–1187. doi: 10.1021/bi00508a020. [DOI] [PubMed] [Google Scholar]
  34. Wallimann T., Szent-Györgyi A. G. An immunological approach to myosin light-chain function in thick filament linked regulation. 2. Effects of anti-scallop myosin light-chain antibodies. Possible regulatory role for the essential light chain. Biochemistry. 1981 Mar 3;20(5):1188–1197. doi: 10.1021/bi00508a021. [DOI] [PubMed] [Google Scholar]
  35. Wallimann T., Turner D. C., Eppenberger H. M. Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle. J Cell Biol. 1977 Nov;75(2 Pt 1):297–317. doi: 10.1083/jcb.75.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]

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