Advertisement

Neurochemical Research

, Volume 20, Issue 4, pp 421–426 | Cite as

Increased susceptibility to degradation by trypsin and subtilisin of in vitro peroxidized myelin proteins

  • E. R. Bongarzone
  • E. F. Soto
  • J. M. Pasquini
Original Articles

Abstract

We examined the possibility that the peroxidative damage to central nervous system myelin produced by reactive oxygen species (ROS), could modify the susceptibility of its proteins to the proteolytic action of proteases such as trypsin and subtilisin. Purified myelin membranes obtained from adult rat brains were “in vitro” peroxidized by two non-enzymatic systems: Fe3+ plus ascorbic acid and Cu2+ plus hydrogen peroxide. Myelin proteins were severely affected by peroxidation. There was an increase in the amount of carbonyl groups (CO), accompanied by and enhanced susceptibility to degradation by trypsin and subtilisin of myelin basic proteins (MBP) and of the major proteolipid protein (PLP). The effect upon the degradation of myelin protein is a possible consequence of the appearance in the structure of myelin proteins of peroxidative modifications that contribute to the recognition by proteolytic enzymes. This hypothesis is supported by the fact that if peroxidation of myelin membranes is done in the presence of EDTA, both CO formation and increased sensitivity to enzymatic breakdown disappear. These results suggest that the appearance of abnormal post-translational modifications in the myelin membrane produced by peroxidation could constitute a putative mechanism of modulating the capacity of myelin proteins to be metabolized by proteases.

Key Words

Oxidative damage peroxidation myelin proteins reactive oxygen species myelin degradation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Banik, N. L., Chakrabarti, A. K., and Hogan, E. L. 1987. Distribution of calcium activated neutral proteinase (mM CANP) in purified myelin and cytosolic fractions of bovine brain white matter. Life Sci. 41:1089–1093.Google Scholar
  2. 2.
    Banik, N. L., Gohil, K., and Davison, A. N. 1976. The action of snake venom, phospholipase A and trypsin on purified myelin in vitro. Biochem. J. 159:273–276.Google Scholar
  3. 3.
    Banik, N. L., McAlhaney, W. W., and Hogan, E. L. 1985. Calcium stimulated proteolysis in myelin: evidence for a Ca2+ activated neutral proteinase associated with purified myelin of rat CNS. J. Neurochem. 45:581–585.Google Scholar
  4. 4.
    Benedetti, A., Compori, M., and Esterbauer, H. 1980. Identification of 4-hydroxynonenal as cytotoxic product originating from the peroxidation of liver microsomal lipids. Biochim. Biophys. Acta 620:281–296.Google Scholar
  5. 5.
    Berlet, H. H., Ilzenhofer, H., and Schulz, G. 1984. Cleavage of myelin basic protein by neutral protease activity of human white matter myelin. J. Neurochem. 43:627–632.Google Scholar
  6. 6.
    Bongarzone, E. R., Pasquini, J. M., and Soto, E. F. 1994. Oxidative damage to proteins and lipids of CNS myelin produced by in vitro generated reactive oxygen species. J. Neurosc. Res. in press.Google Scholar
  7. 7.
    Chantry, A. C., Earl, C., Groome, N., and Glynn, P. 1988. Metalloendoprotease cleavage of 18.2 and 14.1 kilodalton basic proteins dissociating from rodent myelin membranes generates 10.0 and 5.9 kilodalton C-terminal fragments. J. Neurochem. 5:688–692.Google Scholar
  8. 8.
    Connor, J. R., and Fine, R. E. 1987. Development of transferrin-positive oligodendrocytes in the rat central nervous system. J. Neurosc. Res. 17:51–59.Google Scholar
  9. 9.
    Connor, J. R., Menzies, S. L., St Martin, S. M., and Mufson, E. J. 1990. Cellular distribution of transferrin, ferritin and iron in normal and aged human brains. J. Neurosci. Res. 27:595–611.Google Scholar
  10. 10.
    Davies, K. J. A. 1987. Protein damage and degradation by oxygen radicals I. General aspects. J. Biol. Chem. 262:9895–9901.Google Scholar
  11. 11.
    Davies, K. J. A., and Goldberg, A. L. 1987. Proteins damaged by oxygen radicals are rapidly degraded in extracts od red blood cells. J. Biol. Chem. 262:8227–8234.Google Scholar
  12. 12.
    Fucci, L., Oliver, C. N., Coon, M. J., and Stadtman, E. R. 1983. Inactivation of key metabolic enzymes by mixed function oxidation reactions: Possible implication in protein turnover and ageing. Proc. Natl. Acad. Sci. USA. 80:1521–1525.Google Scholar
  13. 13.
    Gerber, M. R., and Connor, J. R. 1989. Do oligodendrocytes mediate iron regulation in the human brain? Neurol. 26:95–98.Google Scholar
  14. 14.
    Halliwell, B., and Gutteridge, J. M. C. 1989. Free radicals in Biology and Medicine. Clarendon press, Oxford.Google Scholar
  15. 15.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.Google Scholar
  16. 16.
    Levine, R. L. 1989. Proteolysis induced by metal catalyzed oxidation. Cell Biol. Rev. 21:347–360.Google Scholar
  17. 17.
    Levine, R. L. 1983. Oxidative modification of glutammine synthase II. Characterization of the ascorbate model system. J. Biol. Chem. 258:11828–11833.Google Scholar
  18. 18.
    Lowry, O. H., Rosebrough N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 250:1099–1105.Google Scholar
  19. 19.
    Murphy, M. E., and Kehrer, J. P. 1989. Oxidation state of tissue thiol groups and content of protein carbonyl groups in chickens with inherited muscular dystrophy. Biochem. J. 260:359–364.Google Scholar
  20. 20.
    Mickel, H. S. 1985. Biological effects of lipid peroxides: lipid peroxidation hypothesis of the etiology of Multiple Sclerosis. The pharmacological effect of lipids II, 215–246.Google Scholar
  21. 21.
    Norton, W. T., and Poduslo, S. E. 1973. Myelination in rat brain: method of myelin isolation. J. Neurochem. 21:749–757.Google Scholar
  22. 22.
    Ohkawa, H., Ohishi, N., and Yagi, K. 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analyt. Biochem. 95:351–358.Google Scholar
  23. 23.
    Schaich, K. M. 1992. Metals and lipid oxidation. Contemporary issues. Lipids 27:209–218.Google Scholar
  24. 24.
    Stadman, E. R. 1990. Metal ion catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free rad. Biol. Med. 9:315–325.Google Scholar
  25. 25.
    Stadman, E. R. 1990. Covalent modification reactions are marking steps in protein turnover. Biochem. 29:6323–6331.Google Scholar
  26. 26.
    Stadman, E. R. 1992. Protein oxidation and aging. Science 257: 1220–1224.Google Scholar
  27. 27.
    Toshiwal, P. K., and Zarling, E. J. 1992. Evidence for increased lipid peroxidation in multiple sclerosis. Neurochem. Res. 17:205–207.Google Scholar
  28. 28.
    Whitaker, J. N. 1987. The presence of immunoreactive myelin basic protein peptide in urine of persons with multiple sclerosis. Ann. Neurol. 22:648–655.Google Scholar
  29. 29.
    Wolff, S. P., and Dean, R. T. 1988. Aldehydes and dicarbonyls in nonenzymic glycosylation of proteins. A rebuttal to Harding and Beswick. Biochem. J. 249:618–619.Google Scholar
  30. 30.
    Wood, J. G., Dawson, R. M. C., and Hauser, H. 1974. Effect of proteolytic attack on the structure of CNS myelin membrane. J. Neurochem. 22:637–642.Google Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • E. R. Bongarzone
    • 1
  • E. F. Soto
    • 1
  • J. M. Pasquini
    • 1
  1. 1.Departamento de Química Biológica, Facultad de Farmacia y Bioquímica e Instituto de Química y Fisicoquímica Biológicas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Universidad de Buenos AiresBuenos AiresArgentina

Personalised recommendations