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. 1983 Nov 1;215(2):227–237. doi: 10.1042/bj2150227

Neurofilaments from ox spinal nerves. Isolation, disassembly, reassembly and cross-linking properties.

M J Carden, P A Eagles
PMCID: PMC1152390  PMID: 6418138

Abstract

An isolation procedure for neurofilaments from ox spinal nerves is described where the triplet polypeptides (which have molecular weights of 205 000, 158 000 and 72 000) constitute more than 80% of the preparation. Soon after purification, the neurofilaments form a gel that is stable for many weeks. The purified neurofilaments disassemble in low-salt buffers at pH greater than 7.0 into soluble particles that contain all of the triplet polypeptides. Greater than 90% of the protein can reassemble to form filaments. The thiol-containing residues in the filaments can be cross-linked. Analyses of the complexes formed show that in the filament the 205 000-mol.wt. components are arranged to that they can be cross-linked to themselves and to the 158 000-mol.wt. polypeptides, and that the 72 000-mol.wt. components are arranged so that their thiol groups can be cross-linked together.

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Selected References

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

  1. Bramhall S., Noack N., Wu M., Loewenberg J. R. A simple colorimetric method for determination of protein. Anal Biochem. 1969 Oct 1;31(1):146–148. doi: 10.1016/0003-2697(69)90251-6. [DOI] [PubMed] [Google Scholar]
  2. Chin T. K., Eagles P. A., Maggs A. The proteolytic digestion of ox neurofilaments with trypsin and alpha-chymotrypsin. Biochem J. 1983 Nov 1;215(2):239–252. doi: 10.1042/bj2150239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chiu F. C., Norton W. T. Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid compositions. J Neurochem. 1982 Nov;39(5):1252–1260. doi: 10.1111/j.1471-4159.1982.tb12562.x. [DOI] [PubMed] [Google Scholar]
  4. Czosnek H., Soifer D., Wisniewski H. M. Heterogeneity of intermediate filament proteins from rabbit spinal cord. Neurochem Res. 1980 Jul;5(7):777–793. doi: 10.1007/BF00964715. [DOI] [PubMed] [Google Scholar]
  5. Delacourte A., Filliatreau G., Boutteau F., Biserte G., Schrevel J. Study of the 10-nm-filament fraction isolated during the standard microtubule preparation. Biochem J. 1980 Nov 1;191(2):543–546. doi: 10.1042/bj1910543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eagles P. A., Gilbert D. S., Maggs A. Neurofilament structure and enzymic modification. Biochem Soc Trans. 1980 Oct;8(5):484–487. doi: 10.1042/bst0080484. [DOI] [PubMed] [Google Scholar]
  7. Eagles P. A., Gilbert D. S., Maggs A. The polypeptide composition of axoplasm and of neurofilaments from the marine worm Myxicola infundibulum. Biochem J. 1981 Oct 1;199(1):89–100. doi: 10.1042/bj1990089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elliott A., Offer G. Shape and flexibility of the myosin molecule. J Mol Biol. 1978 Aug 25;123(4):505–519. doi: 10.1016/0022-2836(78)90204-8. [DOI] [PubMed] [Google Scholar]
  9. Geisler N., Plessmann U., Weber K. Related amino acid sequences in neurofilaments and non-neural intermediate filaments. Nature. 1982 Apr 1;296(5856):448–450. doi: 10.1038/296448a0. [DOI] [PubMed] [Google Scholar]
  10. Geisler N., Weber K. Self-assembly in Vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J Mol Biol. 1981 Sep 25;151(3):565–571. doi: 10.1016/0022-2836(81)90011-5. [DOI] [PubMed] [Google Scholar]
  11. Hogue-Angeletti R. A., Wu H. L., Schlaepfer W. W. Preparative separation and amino acid composition of neurofilament triplet proteins. J Neurochem. 1982 Jan;38(1):116–120. doi: 10.1111/j.1471-4159.1982.tb10861.x. [DOI] [PubMed] [Google Scholar]
  12. Jones S. M., Williams R. C., Jr Phosphate content of mammalian neurofilaments. J Biol Chem. 1982 Sep 10;257(17):9902–9905. [PubMed] [Google Scholar]
  13. Julien J. P., Mushynski W. E. Multiple phosphorylation sites in mammalian neurofilament polypeptides. J Biol Chem. 1982 Sep 10;257(17):10467–10470. [PubMed] [Google Scholar]
  14. Kobashi K. Catalytic oxidation of sulfhydryl groups by o-phenanthroline copper complex. Biochim Biophys Acta. 1968 May;158(2):239–245. doi: 10.1016/0304-4165(68)90136-0. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Liem R. K., Hutchison S. B. Purification of individual components of the neurofilament triplet: filament assembly from the 70 000-dalton subunit. Biochemistry. 1982 Jun 22;21(13):3221–3226. doi: 10.1021/bi00256a029. [DOI] [PubMed] [Google Scholar]
  17. Liem R. K., Yen S. H., Salomon G. D., Shelanski M. L. Intermediate filaments in nervous tissues. J Cell Biol. 1978 Dec;79(3):637–645. doi: 10.1083/jcb.79.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  19. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  20. Quinlan R. A., Franke W. W. Heteropolymer filaments of vimentin and desmin in vascular smooth muscle tissue and cultured baby hamster kidney cells demonstrated by chemical crosslinking. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3452–3456. doi: 10.1073/pnas.79.11.3452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Runge M. S., Schlaepfer W. W., Williams R. C., Jr Isolation and characterization of neurofilaments from mammalian brain. Biochemistry. 1981 Jan 6;20(1):170–175. doi: 10.1021/bi00504a028. [DOI] [PubMed] [Google Scholar]
  22. Schlaepfer W. W., Freeman L. A. Neurofilament proteins of rat peripheral nerve and spinal cord. J Cell Biol. 1978 Sep;78(3):653–662. doi: 10.1083/jcb.78.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schlaepfer W. W. Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve. J Cell Biol. 1977 Jul;74(1):226–240. doi: 10.1083/jcb.74.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schlaepfer W. W. Observations on the disassembly of isolated mammalian neurofilaments. J Cell Biol. 1978 Jan;76(1):50–56. doi: 10.1083/jcb.76.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schlaepfer W. W. Stabilization of neurofilaments by vincristine sulfate in low ionic strength media. J Ultrastruct Res. 1971 Aug;36(3):367–374. doi: 10.1016/s0022-5320(71)80110-7. [DOI] [PubMed] [Google Scholar]
  26. Sharp G. A., Shaw G., Weber K. Immunoelectronmicroscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurones. Exp Cell Res. 1982 Feb;137(2):403–413. doi: 10.1016/0014-4827(82)90042-8. [DOI] [PubMed] [Google Scholar]
  27. Shecket G., Lasek R. J. Neurofilament protein phosphorylation. Species generality and reaction characteristics. J Biol Chem. 1982 May 10;257(9):4788–4795. [PubMed] [Google Scholar]
  28. Steinert P., Zackroff R., Aynardi-Whitman M., Goldman R. D. Isolation and characterization of intermediate filaments. Methods Cell Biol. 1982;24:399–419. doi: 10.1016/s0091-679x(08)60667-6. [DOI] [PubMed] [Google Scholar]
  29. TOMBS M. P., SOUTER F., MACLAGAN N. F. The spectrophotometric determination of protein at 210 millimicrons. Biochem J. 1959 Sep;73:167–171. doi: 10.1042/bj0730167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thorpe R., Delacourte A., Ayers M., Bullock C., Anderton B. H. The polypeptides of isolated brain 10nm filaments and their association with polymerized tubulin. Biochem J. 1979 Aug 1;181(2):275–284. doi: 10.1042/bj1810275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zackroff R. V., Idler W. W., Steinert P. M., Goldman R. D. In vitro reconstitution of intermediate filaments form mammalian neurofilament triplet polypeptides. Proc Natl Acad Sci U S A. 1982 Feb;79(3):754–757. doi: 10.1073/pnas.79.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]

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