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. 1977 Nov 1;75(2):297–317. doi: 10.1083/jcb.75.2.297

Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle

PMCID: PMC2109934  PMID: 264112

Abstract

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.

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Selected References

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  1. Bendall J. R. The steady state kinetic constants of the Mg-activated myofibrillar ATPase. FEBS Lett. 1972 May 15;22(3):330–334. doi: 10.1016/0014-5793(72)80263-1. [DOI] [PubMed] [Google Scholar]
  2. Bessman S. P., Fonyo A. The possible role of the mitochondrial bound creatine kinase in regulation of mitochondrial respiration. Biochem Biophys Res Commun. 1966 Mar 8;22(5):597–602. doi: 10.1016/0006-291x(66)90317-2. [DOI] [PubMed] [Google Scholar]
  3. Blethen S. L., Kaplan N. O. Purification of arginine kinase from lobster and a study of some factors affecting its reactivation. Biochemistry. 1967 May;6(5):1413–1421. doi: 10.1021/bi00857a025. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Botts J., Stone M. J. Kinetics of coupled enzymes. Creatine kinase and myosin A. Biochemistry. 1968 Jul;7(7):2688–2696. doi: 10.1021/bi00847a036. [DOI] [PubMed] [Google Scholar]
  6. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  7. Dawson D. M., Eppenberger H. M., Kaplan N. O. The comparative enzymology of creatine kinases. II. Physical and chemical properties. J Biol Chem. 1967 Jan 25;242(2):210–217. [PubMed] [Google Scholar]
  8. EPPENBERGER H. M., EPPENBERGER M., RICHTERICH R., AEBI H. THE ONTOGENY OF CREATINE KINASE ISOZYMES. Dev Biol. 1964 Aug;10:1–16. doi: 10.1016/0012-1606(64)90002-8. [DOI] [PubMed] [Google Scholar]
  9. Eaton B. L., Pepe F. A. M band protein. Two components isolated from chicken breast muscle. J Cell Biol. 1972 Dec;55(3):681–695. doi: 10.1083/jcb.55.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eppenberger H. M., Dawson D. M., Kaplan N. O. The comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissues. J Biol Chem. 1967 Jan 25;242(2):204–209. [PubMed] [Google Scholar]
  11. 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]
  12. Farrell E. C., Jr, Baba N., Brierley G. P., Grümer H. D. On the creatine phosphokinase of heart muscle mitochondria. Lab Invest. 1972 Aug;27(2):209–213. [PubMed] [Google Scholar]
  13. Fitch C. D., Jellinek M., Mueller E. J. Experimental depletion of creatine and phosphocreatine from skeletal muscle. J Biol Chem. 1974 Feb 25;249(4):1060–1063. [PubMed] [Google Scholar]
  14. Gercken G., Schlette U. Metabolite status of the heart in acute insufficiency due to 1-fluoro-2,4-dinitrobenzene. Experientia. 1968 Jan 15;24(1):17–19. doi: 10.1007/BF02136764. [DOI] [PubMed] [Google Scholar]
  15. Gudbjarnason S., Mathes P., Ravens K. G. Functional compartmentation of ATP and creatine phosphate in heart muscle. J Mol Cell Cardiol. 1970 Sep;1(3):325–339. doi: 10.1016/0022-2828(70)90009-x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. JOBSIS F. F. Early kinetics of the cytochrome B response to muscular contraction. Ann N Y Acad Sci. 1959 Aug 28;81:505–509. doi: 10.1111/j.1749-6632.1959.tb49331.x. [DOI] [PubMed] [Google Scholar]
  18. Jacobs H., Heldt H. W., Klingenberg M. High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase. Biochem Biophys Res Commun. 1964 Aug 11;16(6):516–521. doi: 10.1016/0006-291x(64)90185-8. [DOI] [PubMed] [Google Scholar]
  19. Jacobus W. E., Lehninger A. L. Creatine kinase of rat heart mitochondria. Coupling of creatine phosphorylation to electron transport. J Biol Chem. 1973 Jul 10;248(13):4803–4810. [PubMed] [Google Scholar]
  20. KUBY S. A., NODA L., LARDY H. A. Adenosinetriphosphate-creatine transphosphorylase. III. Kinetic studies. J Biol Chem. 1954 Sep;210(1):65–82. [PubMed] [Google Scholar]
  21. Khan M. A., Holt P. G., Knight J. O., Kakulas B. A. Incubation film technique for the histochemical localization of creatine kinase. Histochemie. 1971;26(2):120–125. doi: 10.1007/BF00293502. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kundrat E., Pepe F. A. The M band. Studies with fluorescent antibody staining. J Cell Biol. 1971 Feb;48(2):340–347. doi: 10.1083/jcb.48.2.340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. MacGillivray A. J., Cameron A., Krauze R. J., Rickwood D., Paul J. The non-histone proteins of chromatin, their isolation and composition in a number of tissues. Biochim Biophys Acta. 1972 Aug 25;277(2):384–402. [PubMed] [Google Scholar]
  27. 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]
  28. Masaki T., Takaiti O. M-protein. J Biochem. 1974 Feb;75(2):367–380. doi: 10.1093/oxfordjournals.jbchem.a130403. [DOI] [PubMed] [Google Scholar]
  29. McGilvery R. W., Murray T. W. Calculated equilibria of phosphocreatine and adenosine phosphates during utilization of high energy phosphate by muscle. J Biol Chem. 1974 Sep 25;249(18):5845–5850. [PubMed] [Google Scholar]
  30. Moore C. H., Farron F., Bohnert D., Weissmann C. Possible origin of a minor virus specific protein (A1) in Q-beta particles. Nat New Biol. 1971 Sep 15;234(50):204–206. doi: 10.1038/newbio234204a0. [DOI] [PubMed] [Google Scholar]
  31. Moos C., Eisenberg E., Estes J. E. Bound-nucleotide exchange in actin and actomyosin. Biochim Biophys Acta. 1967 Dec 12;147(3):536–545. doi: 10.1016/0005-2795(67)90013-x. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Nägle S. Die Bedeutung von Kreatinphosphat und Adenosintriphosphat im Hinblick auf Energiebereitstellung, -transport und -verwertung im normalen und insuffizienten Herzmuskel. Klin Wochenschr. 1970 Mar 15;48(6):332–341. doi: 10.1007/BF01484859. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Pepe F. A. Some aspects of the structural organization of the myofibril as revealed by antibody--staining methods. J Cell Biol. 1966 Mar;28(3):505–525. doi: 10.1083/jcb.28.3.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pepe F. A. Structure of muscle filaments from immunohistochemical and ultrastructural studies. J Histochem Cytochem. 1975 Jul;23(7):543–562. doi: 10.1177/23.7.1095653. [DOI] [PubMed] [Google Scholar]
  37. Pepe F. A. Structure of muscle filaments from immunohistochemical and ultrastructural studies. J Histochem Cytochem. 1975 Jul;23(7):543–562. doi: 10.1177/23.7.1095653. [DOI] [PubMed] [Google Scholar]
  38. Saks V. A., Chernousova G. B., Voronkov I. I., Smirnov V. N., Chazov E. I. Study of energy transport mechanism in myocardial cells. Circ Res. 1974 Sep;35 (Suppl 3):138–149. [PubMed] [Google Scholar]
  39. Sherwin A. L., Karpati G., Bulcke J. A. Immunohistochemical localization of creatine phosphokinase in skeletal muscle. Proc Natl Acad Sci U S A. 1969 Sep;64(1):171–175. doi: 10.1073/pnas.64.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Thornell L. E., Sjöström M. The myofibrillar M-band in the cryo-section-analysis of section thickness. J Microsc. 1975 Aug;104(3):263–269. doi: 10.1111/j.1365-2818.1975.tb04024.x. [DOI] [PubMed] [Google Scholar]
  41. Tonomura Y., Appel P., Morales M. On the molecular weight of myosin. II. Biochemistry. 1966 Feb;5(2):515–521. doi: 10.1021/bi00866a017. [DOI] [PubMed] [Google Scholar]
  42. Turner D. C., Gmür R., Lebherz H. G., Siegrist M., Wallimann T., Eppenberger H. M. Differentiation in cultures derived from embryonic chicken muscle. II. Phosphorylase histochemistry and fluorescent antibody staining for creatin kinase and aldolase. Dev Biol. 1976 Feb;48(2):284–307. doi: 10.1016/0012-1606(76)90091-9. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Yagi K., Nakata T., Yazawa Y., Sakakibara I. Rate determining step of the coupled reaction system composed of H-meromyosin-adenosinetriphosphatase and creatine kinase. J Biochem. 1965 Jun;57(6):766–772. doi: 10.1093/oxfordjournals.jbchem.a128143. [DOI] [PubMed] [Google Scholar]

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