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Heterogeneity of intermediate filament proteins from rabbit spinal cord

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Abstract

Large amounts of a neurofilament-enriched fraction may be prepared from spinal cord homogenates by a simple, three-step procedure. This involves flotation of filament-containing axon fragments, extraction with Triton X-100, and washing by sedimentation through a sucrose density gradient. The material obtained by this procedure includes both large mats of individual 10-nm filaments and tightly packed bundles of filaments. SDS-gel electrophoresis of these fractions indicates that the fractions are formed of four polypeptides: the three which are generally considered to form neurofilaments (P200, P150, and P68) and another, with a molecular weight of about 50,000 daltons (P50), which is thought to be derived from fibrous astrocytes. Analysis of these filament fractions on two-dimensional gels indicates heterogeneity among each of the different molecular weight classes. The largest polypeptide of neurofilaments, P200, focuses at several spots in the pH gradient. P68 and P150 are more acidic: each appears as a pair of overlapping spots. P50 resolves into a complex of spots of about the same molecular weight but with different isoelectric points. Heterogeneity is not unique to these filament polypeptides but appears to be a characteristic of all fibrous proteins of the nervous system.

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References

  1. Marotta, C. A., Strocchi, P., andGilbert, J. M. 1978. Microheterogeneity of brain cytoplasmic and synaptoplasmic actins. J. Neurochem. 30:1441–1451.

    PubMed  Google Scholar 

  2. Feit, H. Neudeck, U., andBaskin, F. 1977. Comparison of the isoelectric and molecular weight properties of tubulin subunits. J. Neurochem. 28:697–706.

    PubMed  Google Scholar 

  3. Lu, R. C., andElzinga, M. 1977. Chromatographic resolution of the subunits of calf brain tubulin. Anal. Biochem. 77:243–250.

    PubMed  Google Scholar 

  4. Gozes, I., andLittauer, U. Z. 1978. Tubulin microheterogeneity increases with rat brain maturation. Nature 276:411–413.

    PubMed  Google Scholar 

  5. Marotta, C. A., Harris, J. L., andGilbert, J. M. 1978. Characterization of multiple forms of brain tubulin subunits. J. Neurochem. 30:1431–1440.

    PubMed  Google Scholar 

  6. Dahl, J. L., andWeibel, V. J. 1979 Changes in tubulin heterogeneity during postnatal development of rat brain. Biochem. Biophys. Res. Commun. 86:822–828.

    PubMed  Google Scholar 

  7. Shelanski, N. L., andFeit, H. 1972. Filaments and tubules in the nervous system. Pages 47–80,in Bourne, G. H. (ed.), The Structure and Function of Nervous Tissue, Vol. 6, Academic Press, New York.

    Google Scholar 

  8. Wuerker, R. B., andKirkpatrick, J. B. 1972. Neuronal microtubules, neurofilaments and microfilaments. Int. Rev. Cytol. 33:45–75.

    PubMed  Google Scholar 

  9. Hoffman, P. N., andLasek, R. J. 1975. The slow component of axonal transport. Identification of major structural polypeptides of axon and their generality among mammalian neurons. J. Cell Biol. 66:351–366.

    PubMed  Google Scholar 

  10. Schlaepfer, W. W., andFreeman, L. 1978. Neurofilament proteins of rat peripheral nerve and spinal cord. J. Cell Biol. 78:653–662.

    PubMed  Google Scholar 

  11. Liem, R. K. H., Yen, S. N., Salomon, G. D., andShelanski, M. L. 1978. Intermediate filaments in nervous tissues. J. Cell Biol. 79:637–645.

    PubMed  Google Scholar 

  12. Eng, L. F., Vanderhaeghen, J. J., Bignami, A., andGerstl, B. 1971. An acidic protein isolated from fibrous astrocytes. Brain Res. 28:351–354.

    PubMed  Google Scholar 

  13. Goldman, J. E., Schaumburg, H. H., andNorton, W. T. 1978. Isolation and characterization of glial filaments from human brain. J. Cell Biol. 78:426–440.

    PubMed  Google Scholar 

  14. Shelanski, M. L., Albert, S., DeVries, G. H., andNorton, W. T. 1971. Isolation of filaments from brain. Science 174:1242–1245.

    PubMed  Google Scholar 

  15. DeVries, G. H., Norton, W. T., andRaine, C. S. 1972. Axons: Isolation from mammalian central nervous system. Science 175:1370–1372.

    PubMed  Google Scholar 

  16. Yen, S.-H., Dahl, D., Schachner, M., andShelanski, M. 1976. Biochemistry of the filaments from brain. Proc. Natl. Acad. Sci. U.S.A. 73:529–533.

    PubMed  Google Scholar 

  17. Davison, P. F., andHong, B.-S. 1977. Structural homologies in mammalian neurofilament proteins. Brain Res. 134:287–295.

    PubMed  Google Scholar 

  18. Thorpe, R., Delacourte, A., andAnderton, B. H. 1979. The isolation of brain 10 nm filaments polypeptides from urea extracts of brain white matter. FEBS Lett. 103:148–151.

    PubMed  Google Scholar 

  19. Schlaepfer, W. W. 1977. Studies on the isolation and substructure of mammalian neurofilaments. J. Ultrastruct. Res. 76:50–56.

    Google Scholar 

  20. Runge, M. S., Detrich, H. W., III, andWilliams, R. C., Jr. 1979. Identification of the major 68,000-dalton protein of microtubule preparations as a 10-nm filament protein and its effect on microtubule assembly in vitro. Biochemistry 18:1689–1698.

    PubMed  Google Scholar 

  21. Czosnek, H., Soifer, D., Hochberg, A., andWisniewski, H. M. 1979. Isolation and characterization of free and membrane-bound polyribosomes from rabbit spinal cord. J. Neurosci. Methods 1:327–341.

    PubMed  Google Scholar 

  22. Czosnek, H., Soifer, D., andWisniewski, H. M. 1980. Studies on the biosynthesis of neurofilament proteins. J. Cell Biol. 85:726–734.

    PubMed  Google Scholar 

  23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

    PubMed  Google Scholar 

  24. O'Farrell, P. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007–4021.

    PubMed  Google Scholar 

  25. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.

    PubMed  Google Scholar 

  26. Folch, J., Lees, M., andSloane-Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–509.

    PubMed  Google Scholar 

  27. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466–468.

    PubMed  Google Scholar 

  28. Bloemendal, H., Bont, W. S., DeVries, M., andBenedetti, E. L. 1967. Isolation and properties of polyribosomes and fragments of the endoplasmic reticulum from rat liver. Biochem. J. 103:177–182.

    PubMed  Google Scholar 

  29. Spurr, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31–43.

    PubMed  Google Scholar 

  30. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208–212.

    PubMed  Google Scholar 

  31. Benitz, W. E., Dahl, D., Williams, K. W., andBignami, A. 1976. The protein composition of glial and nerve fibers. FEBS Lett. 66:285–289.

    PubMed  Google Scholar 

  32. DeVries, G. H., Eng, L. F., Lewis, D. L., andHadfield, M. G. 1976. The protein composition of bovine myelin-free axons. Biochim. Biophys. Acta 439:133–145.

    PubMed  Google Scholar 

  33. Anderton, B. H., Ayers, M., andThorpe, R. 1978. Neurofilaments from mammalian central and peripheral nerve share certain polypeptides. FEBS Lett. 96:159–163.

    PubMed  Google Scholar 

  34. Dahl, D. 1979. The cyanogen bromide peptide maps of neurofilament polypeptides in axonal preparations isolated from bovine brain are different. FEBS Lett. 103:144–147.

    PubMed  Google Scholar 

  35. Wang, K., Ash, J. F., andSinger, S. J. 1975. Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells. Proc. Natl. Acad. Sci. U.S.A. 72:483–486.

    PubMed  Google Scholar 

  36. Czosnek, H., Soifer, D., andWisniewski, H. M. 1979. Are the triplet polypeptides of neurofilaments formed by proteolytic degradation? J. Cell Biol. 83 (2pt II). 478a (abstract).

    Google Scholar 

  37. Marotta, C. A., Strocchi, P., andGilbert, J. M. 1979. Biosynthesis of heterogeneous forms of mammalian brain tubulin subunits by multiple messenger RNAs. J. Neurochem. 33:231–246.

    PubMed  Google Scholar 

  38. Willard, M. B. 1976. A genetically-determined protein polymorphism in the rabbit nervous system. Proc. Natl. Acad. Sci. U.S.A. 73:3641–3645.

    PubMed  Google Scholar 

  39. Soifer, D., Iqbal, K., De Martini, J., Sturman, J. A., andWisniewski, H. M. 1980. Protein changes associated with Wallerian degeneration. Trans. Am. Soc. Neurochem. 11:79 (abstract).

    Google Scholar 

  40. Forgue, S. T., andDahl, J. L. 1979. Rat brain tubulin: subunit heterogeneity and phosphorylation. J. Neurochem. 32:1015–1025.

    PubMed  Google Scholar 

  41. Scheket, G., andLasek, R. J. 1979. Phosphorylation of neurofilaments from mammalian peripheral nerve. Trans. Am. Soc. Neurochem. 10:140 (abstract).

    Google Scholar 

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Authors and Affiliations

  1. Department of Pathological Neurobiology, Institute for Basic Research in Mental Retardation, 10314, Staten Island, New York

    Henryk Czosnek, David Soifer & Henryk M. Wisniewski

  2. Department of Anatomy and Cell Biology Downstate Medical Center, SUNY, 11203, Brooklyn, New York

    David Soifer

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  1. Henryk Czosnek
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  2. David Soifer
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  3. Henryk M. Wisniewski
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Czosnek, H., Soifer, D. & Wisniewski, H.M. Heterogeneity of intermediate filament proteins from rabbit spinal cord. Neurochem Res 5, 777–793 (1980). https://doi.org/10.1007/BF00964715

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