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. 1982 Jan 1;201(1):39–47. doi: 10.1042/bj2010039

Phosphorylation in vivo of four basic proteins of rat brain myelin

Harish C Agrawal 1, Keith O'Connell 1, Charlotte L Randle 1, Daya Agrawal 1
PMCID: PMC1163607  PMID: 7082288

Abstract

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H332PO4. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [32P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.

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Selected References

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

  1. Agrawal H. C., Burton R. M., Fishman M. A., Mitchell R. F., Prensky A. L. Partial characterization of a new myelin protein component. J Neurochem. 1972 Sep;19(9):2083–2089. doi: 10.1111/j.1471-4159.1972.tb05118.x. [DOI] [PubMed] [Google Scholar]
  2. Agrawal H. C., Hartman B. K., Shearer W. T., Kalmbach S., Margolis F. L. Purification and immunohistochemical localization of rat brain myelin proteolipid protein. J Neurochem. 1977 Mar;28(3):495–508. doi: 10.1111/j.1471-4159.1977.tb10420.x. [DOI] [PubMed] [Google Scholar]
  3. Agrawal H. C., Trotter J. L., Burton R. M., Mitchell R. F. Metabolic studies on myelin. Evidence for a precursor role of a myelin subfraction. Biochem J. 1974 Apr;140(1):99–109. doi: 10.1042/bj1400099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allerton S. E., Perlmann G. E. Chemical characterization of the phosphoprotein phosvitin. J Biol Chem. 1965 Oct;240(10):3892–3898. [PubMed] [Google Scholar]
  5. Allison J. H., Agrawal H. C., Moore B. W. Effect of N,N,N',N'-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels. Anal Biochem. 1974 Apr;58(2):592–601. doi: 10.1016/0003-2697(74)90228-0. [DOI] [PubMed] [Google Scholar]
  6. Barbarese E., Carson J. H., Braun P. E. Accumulation of the four myelin basic proteins in mouse brain during development. J Neurochem. 1978 Oct;31(4):779–782. doi: 10.1111/j.1471-4159.1978.tb00110.x. [DOI] [PubMed] [Google Scholar]
  7. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  8. Carnegie P. R., Dunkley P. R., Kemp B. E., Murray A. W. Phosphorylation of selected serine and threonine residues in myelin basic protein by endogenous and exogenous protein kinases. Nature. 1974 May 10;249(453):147–150. doi: 10.1038/249147a0. [DOI] [PubMed] [Google Scholar]
  9. Carnegie P. R., Kemp B. E., Dunkley P. R., Murray A. W. Phosphorylation of myelin basic protein by an adenosine 3':5'-cyclic monophosphate-dependent protein kinase. Biochem J. 1973 Nov;135(3):569–572. doi: 10.1042/bj1350569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunkley P. R., Carnegie P. R. Amino acid sequence of the smaller basic protein from rat brain myelin. Biochem J. 1974 Jul;141(1):243–255. doi: 10.1042/bj1410243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fishman M. A., Hagen S., Trotter J. L., O'Connell K., Agrawal H. C. Use of a stable fluorescent reagent, 2-methoxy-2,4-diphenyl-3(2H)-furanone, for the visualization and purification of myelin proteins. J Neurochem. 1979 Mar;32(3):1077–1083. doi: 10.1111/j.1471-4159.1979.tb04596.x. [DOI] [PubMed] [Google Scholar]
  12. Golds E. E., Braun P. E. Cross-linking studies on the conformation and dimerization of myelin basic protein in solution. J Biol Chem. 1978 Nov 25;253(22):8171–8177. [PubMed] [Google Scholar]
  13. Gonzalez-Sastre F. The protein composition of isolated myelin. J Neurochem. 1970 Jul;17(7):1049–1056. doi: 10.1111/j.1471-4159.1970.tb02258.x. [DOI] [PubMed] [Google Scholar]
  14. Hartman B. K., Agrawal H. C., Kalmbach S., Shearer W. T. A comparative study of the immunohistochemical localization of basic protein to myelin and oligodendrocytes in rat and chicken brain. J Comp Neurol. 1979 Nov 15;188(2):273–290. doi: 10.1002/cne.901880206. [DOI] [PubMed] [Google Scholar]
  15. Martenson R. E., Deibler G. E., Kies M. W. Electrophoretic characterization of basic proteins in acid extracts of central nervous system tissue. J Neurochem. 1971 Dec;18(12):2417–2426. doi: 10.1111/j.1471-4159.1971.tb00197.x. [DOI] [PubMed] [Google Scholar]
  16. Martenson R. E., Deibler G. E., Kies M. W. Extraction of rat myelin basic protein free of other basic proteins of whole central nervous system tissue. An analysis of its electrophoretic heterogeneity. J Biol Chem. 1969 Aug 25;244(16):4268–4272. [PubMed] [Google Scholar]
  17. Martenson R. E., Deibler G. E., Kies M. W., McKneally S. S., Shapira R., Kibler R. F. Differences between the two myelin basic proteins of the rat central nervous system. A deletion in the smaller protein. Biochim Biophys Acta. 1972 Mar 15;263(1):193–203. doi: 10.1016/0005-2795(72)90172-9. [DOI] [PubMed] [Google Scholar]
  18. Martenson R. E., Deibler G. E. Partial characterization of basic proteins of chicken, turtle and frog central nervous system myelin. J Neurochem. 1975 Jan;24(1):79–88. doi: 10.1111/j.1471-4159.1975.tb07631.x. [DOI] [PubMed] [Google Scholar]
  19. Matthieu J. M., Kuffer A. D. In vivo incorporation of 32P into myelin basic protein from normal and quaking mice. Adv Exp Med Biol. 1978;100:159–170. doi: 10.1007/978-1-4684-2514-7_11. [DOI] [PubMed] [Google Scholar]
  20. McNamara J. O., Appel S. H. Myelin basic protein phosphatase activity in rat brain. J Neurochem. 1977 Jul;29(1):27–35. doi: 10.1111/j.1471-4159.1977.tb03920.x. [DOI] [PubMed] [Google Scholar]
  21. Mehl E., Halaris A. Stoichiometric relation of protein components in cerebral myelin from different species. J Neurochem. 1970 May;17(5):659–668. doi: 10.1111/j.1471-4159.1970.tb00545.x. [DOI] [PubMed] [Google Scholar]
  22. Miyamoto E., Kakiuchi S. In vitro and in vivo phosphorylation of myelin basic protein by exogenous and endogenous adenosine 3':5'-monophosphate-dependent protein kinases in brain. J Biol Chem. 1974 May 10;249(9):2769–2777. [PubMed] [Google Scholar]
  23. Miyamoto E., Kakiuchi S. Phosphoprotein phosphatases for myelin basic protein in myelin and cytosol fractions of brain. Biochim Biophys Acta. 1975 Apr 19;384(2):458–465. doi: 10.1016/0005-2744(75)90046-7. [DOI] [PubMed] [Google Scholar]
  24. Miyamoto E. Phosphorylation of endogenous proteins in myelin of rat brain. J Neurochem. 1976 Mar;26(3):573–577. doi: 10.1111/j.1471-4159.1976.tb01513.x. [DOI] [PubMed] [Google Scholar]
  25. Miyamoto E. Protein kinases in myelin of rat brain: solubilization and characterization. J Neurochem. 1975 Mar;24(3):503–512. doi: 10.1111/j.1471-4159.1975.tb07668.x. [DOI] [PubMed] [Google Scholar]
  26. Nussbaum J. L., Rouayrenc J. F., Mandel P., Jollès J., Jollès P. Isolation and terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein. Biochem Biophys Res Commun. 1974 Apr 23;57(4):1240–1247. doi: 10.1016/0006-291x(74)90829-8. [DOI] [PubMed] [Google Scholar]
  27. Petrali E. H., Thiessen B. J., Sulakhe P. V. Characteristics of magnesium-dependent, Ca2+ -stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter. Arch Biochem Biophys. 1980 Dec;205(2):520–535. doi: 10.1016/0003-9861(80)90135-6. [DOI] [PubMed] [Google Scholar]
  28. Randerath K. An evaluation of film detection methods for weak beta-emitters, particularly tritium. Anal Biochem. 1970 Mar;34:188–205. doi: 10.1016/0003-2697(70)90100-4. [DOI] [PubMed] [Google Scholar]
  29. Steck A. J., Appel S. H. Phosphorylation of myelin basic protein. J Biol Chem. 1974 Sep 10;249(17):5416–5420. [PubMed] [Google Scholar]
  30. Sulakhe P. V., Petrali E. H., Davis E. R., Thiessen B. J. Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain. Biochemistry. 1980 Nov 11;19(23):5363–5371. doi: 10.1021/bi00564a034. [DOI] [PubMed] [Google Scholar]
  31. Sulakhe P. V., Petrali E. H., Thiessen B. J., Davis E. R. Calcium ion-stimulated phosphorylation of myelin proteins. Biochem J. 1980 Feb 15;186(2):469–473. doi: 10.1042/bj1860469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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