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. 1985 Apr 1;100(4):1063–1072. doi: 10.1083/jcb.100.4.1063

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

PMCID: PMC2113768  PMID: 3884630

Abstract

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000- 43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.

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Selected References

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  1. Barrantes F. J., Braceras A., Caldironi H. A., Mieskes G., Moser H., Toren E. C., Jr, Roque M. E., Wallimann T., Zechel A. Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases. J Biol Chem. 1985 Mar 10;260(5):3024–3034. [PubMed] [Google Scholar]
  2. Barrantes F. J., Mieskes G., Wallimann T. A membrane-associated creatine kinase (EC 2.7.3.2) identified as an acidic species of the non-receptor, peripheral nu-proteins in Torpedo acetylcholine receptor membranes. FEBS Lett. 1983 Feb 21;152(2):270–276. doi: 10.1016/0014-5793(83)80394-9. [DOI] [PubMed] [Google Scholar]
  3. Barrantes F. J., Mieskes G., Wallimann T. Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5440–5444. doi: 10.1073/pnas.80.17.5440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrantes F. J. Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the Mr 43,000 nonreceptor peptide. J Cell Biol. 1982 Jan;92(1):60–68. doi: 10.1083/jcb.92.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barrantes F. J. Recent developments in the structure and function of the acetylcholine receptor. Int Rev Neurobiol. 1983;24:259–341. doi: 10.1016/s0074-7742(08)60224-x. [DOI] [PubMed] [Google Scholar]
  6. Baskin R. J., Deamer D. W. A membrane-bound creatine phosphokinase in fragmented sarcoplasmic reticulum. J Biol Chem. 1970 Mar 25;245(6):1345–1347. [PubMed] [Google Scholar]
  7. Berl S., Puszkin S., Nicklas W. J. Actomyosin-like protein in brain. Science. 1973 Feb 2;179(4072):441–446. doi: 10.1126/science.179.4072.441. [DOI] [PubMed] [Google Scholar]
  8. Bessman S. P., Geiger P. J. Transport of energy in muscle: the phosphorylcreatine shuttle. Science. 1981 Jan 30;211(4481):448–452. doi: 10.1126/science.6450446. [DOI] [PubMed] [Google Scholar]
  9. Büechi M., Bächi T. Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus. J Cell Biol. 1979 Nov;83(2 Pt 1):338–347. doi: 10.1083/jcb.83.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Cartaud J., Sobel A., Rousselet A., Devaux P. F., Changeux J. P. Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ. J Cell Biol. 1981 Aug;90(2):418–426. doi: 10.1083/jcb.90.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conti-Tronconi B. M., Dunn S. M., Raftery M. A. Functional stability of Torpedo acetylcholine receptor. Effects of protease treatment. Biochemistry. 1982 Mar 2;21(5):893–899. doi: 10.1021/bi00534a013. [DOI] [PubMed] [Google Scholar]
  13. Friedhoff A. J., Lerner M. H. Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles. Life Sci. 1977 Mar 1;20(5):867–873. doi: 10.1016/0024-3205(77)90039-x. [DOI] [PubMed] [Google Scholar]
  14. Gordon A. S., Milfay D., Diamond I. Identification of a molecular weight 43,000 protein kinase in acetylcholine receptor-enriched membranes. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5862–5865. doi: 10.1073/pnas.80.19.5862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grosse R., Spitzer E., Kupriyanov V. V., Saks V. A., Repke K. R. Coordinate interplay between (Na+ + K+)-ATPase and creatine phosphokinase optimizes (Na+/K+)-antiport across the membrane of vesicles formed from the plasma membrane of cardiac muscle cell. Biochim Biophys Acta. 1980 Dec 2;603(1):142–156. doi: 10.1016/0005-2736(80)90397-1. [DOI] [PubMed] [Google Scholar]
  16. Gysin R., Yost B., Flanagan S. D. Immunochemical and molecular differentiation of 43 000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses. Biochemistry. 1983 Dec 6;22(25):5781–5789. doi: 10.1021/bi00294a016. [DOI] [PubMed] [Google Scholar]
  17. Hartig P. R., Raftery M. A. Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes. Biochemistry. 1979 Apr 3;18(7):1146–1150. doi: 10.1021/bi00574a004. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Kistler J., Stroud R. M., Klymkowsky M. W., Lalancette R. A., Fairclough R. H. Structure and function of an acetylcholine receptor. Biophys J. 1982 Jan;37(1):371–383. doi: 10.1016/S0006-3495(82)84685-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levitskii D. O., Levchenko T. S., Saks V. A., Sharov V. G., Smirnov V. N. Funktsional'noe sopriazhenie mezhdu Ca2+-ATPazoi i kreatinfosfokinazoi v sarkoplazmaticheskom retikulume serdechnoi myshtsy. Biokhimiia. 1977 Oct;42(10):1766–1773. [PubMed] [Google Scholar]
  21. Nghiêm H. O., Cartaud J., Dubreuil C., Kordeli C., Buttin G., Changeux J. P. Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6403–6407. doi: 10.1073/pnas.80.20.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Perriard J. C., Caravatti M., Perriard E. R., Eppenberger H. M. Quantitation of creatine kinase isoenzyme transition in differentiating chicken embryonic breast muscle and myogenic cell cultures by immunoadsorption. Arch Biochem Biophys. 1978 Nov;191(1):90–100. doi: 10.1016/0003-9861(78)90070-x. [DOI] [PubMed] [Google Scholar]
  23. Rosenberg U. B., Eppenberger H. M., Perriard J. C. Occurrence of heterogenous forms of the subunits of creatine kinase in various muscle and nonmuscle tissues and their behaviour during myogenesis. Eur J Biochem. 1981 May;116(1):87–92. doi: 10.1111/j.1432-1033.1981.tb05304.x. [DOI] [PubMed] [Google Scholar]
  24. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  25. Saks V. A., Rosenshtraukh L. V., Smirnov V. N., Chazov E. I. Role of creatine phosphokinase in cellular function and metabolism. Can J Physiol Pharmacol. 1978 Oct;56(5):691–706. doi: 10.1139/y78-113. [DOI] [PubMed] [Google Scholar]
  26. Sealock R. Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting. J Cell Biol. 1982 Feb;92(2):514–522. doi: 10.1083/jcb.92.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sharov V. G., Saks V. A., Smirnov V. N., Chazov E. I. An electron microscopic histochemical investigation of the localization of creatine phosphokinase in heart cells. Biochim Biophys Acta. 1977 Aug 1;468(3):495–501. doi: 10.1016/0005-2736(77)90299-1. [DOI] [PubMed] [Google Scholar]
  28. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  29. Turner D. C., Maier V., Eppenberger H. M. Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells. Dev Biol. 1974 Mar;37(1):63–89. doi: 10.1016/0012-1606(74)90170-5. [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., Moser H., Eppenberger H. M. Isoenzyme-specific localization of M-line bound creatine kinase in myogenic cells. J Muscle Res Cell Motil. 1983 Aug;4(4):429–441. doi: 10.1007/BF00711948. [DOI] [PubMed] [Google Scholar]
  32. Wallimann T., Schlösser T., Eppenberger H. M. Function of M-line-bound creatine kinase as intramyofibrillar ATP regenerator at the receiving end of the phosphorylcreatine shuttle in muscle. J Biol Chem. 1984 Apr 25;259(8):5238–5246. [PubMed] [Google Scholar]
  33. 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]
  34. Wennogle L. P., Changeux J. P. Transmembrane orientation of proteins present in acetylcholine receptor-rich membranes from Torpedo marmorata studied by selective proteolysis. Eur J Biochem. 1980 May;106(2):381–393. doi: 10.1111/j.1432-1033.1980.tb04584.x. [DOI] [PubMed] [Google Scholar]
  35. Wildhaber I., Gross H., Moor H. The control of freeze-drying with deuterium oxide (D2O). J Ultrastruct Res. 1982 Sep;80(3):367–373. doi: 10.1016/s0022-5320(82)80050-6. [DOI] [PubMed] [Google Scholar]
  36. Zingsheim H. P., Neugebauer D. C., Frank J., Hänicke W., Barrantes F. J. Dimeric arrangement and structure of the membrane-bound acetylcholine receptor studied by electron microscopy. EMBO J. 1982;1(5):541–547. doi: 10.1002/j.1460-2075.1982.tb01206.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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