Abstract
Axonal remnants are considered a probable source of contamination of isolated myelin in view of the relatively tight axon-glial intercellular junction. Using the rabbit optic system to label specifically axonal components, we have found the levels of such contaminants to depend on the myelin isolation procedure, the tissue source, and the nature of the contaminant. A procedure employing repetitive treatments with EGTA was found to be highly effective in removing proline-labeled axonal proteins, the estimated upper limit of such contamination being approximately 0.6–1.2% of the myelin protein. The standard isolation procedure of Norton and Poduslo, supplemented with an additional discontinuous gradient step, proved equally effective in removing rapidly transported proteins from myelin isolated from the superior colliculus or lateral geniculate body. When the optic tract was the source, however, the EGTA procedure proved more effective in removing both rapidly and slowly transported proteins. Axonal gangliosides labeled with N-[3H]acetylmannosamine were efficiently removed by both procedures, adding support to the proposition that gangliosides detected in isolated myelin are intrinsic to that membrane.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ando, S., Chang, N. -C. and Yu, R. K. (1978). High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species.Anal. Biochem. 89437–450.
August, C., Davison, A. N., and Williams, F. M. (1961). Phospholipid metabolism in nervous tissue.Biochem. J. 818–12.
Autilio, L. A., Norton, W. T., and Terry, R. D. (1964). The preparation and some properties of purified myelin from the central nervous system.J. Neurochem. 1117–27.
Autilio-Gambetti, L., Gambetti, P., and Shafer, B. (1975). Glial and neuronal contribution to proteins and glycoproteins recovered in myelin fractions.Brain Res. 84336–340.
Blank, W. F., Jr., Bartlett-Bunge, M., and Bunge, R. P. (1974). The sensitivity of the myelin sheath, particularly the Schwann cell-axolemmal junction, to lowered calcium levels in cultured sensory ganglia.Brain Res. 67503–518.
DeVries, G. H. (1976). Isolation and axolemma-enriched fractions from bovine central nervous system.Neurosci. Lett. 3117–122.
DeVries, G. H., and Zmachinski, C. J. (1980). The lipid composition of rat CNS axolemma-enriched fractions.J. Neurochem. 34424–430.
DeVries, G. H., Norton, W. T., and Raine, C. S. (1972). Axons: Isolation from mammalian central nervous system.Science 1721370–1372
DeVries, G. H., Matthieu, J. -M., Beny, M., Chicheportiche, R., Lazdunski, M., and Dolivo, M. (1978). Isolation and partial characterization of rat CNS axolemma enriched fractions.Brain Res. 147339–352.
Droz, D., DiGiamberardino, L., Koenig, H. L., Boyenval, J., and Hassig, R. (1978). Axon-myelin transfer of phospholipid components in the course of their axonal transport as visualized by radioautography.Brain Res. 155347–353.
Elam, J. S. (1974), Association of axonally transported proteins with goldfish brain myelin fractions.J. Neurochem. 23345–354.
Elam, J. S. (1975). Association of proteins undergoing slow axonal transport with goldfish visual system myelin.Brain Res. 97303–315.
Elam, J. S. (1978). Dissociation of axonally transported proteins from myelin by ethylenediamine tetraacetate (EDTA).J. Neurochem. 31351–353.
Elam, J. S., and Agranoff, B. W. (1971). Rapid transport of protein in the optic system of the goldfish.J. Neurochem. 18375–387.
Gerstl, B., Rubinstein, L. -J., Eng, L. F., and Tavaststjerna, M. (1966). A neurochemical study of a case of sudanophilic leukodystrophy.Arch. Neurol. 15603–614.
Greenfield, S., Norton, W. T., and Morell, P. (1971). Quaking mouse: Isolation and characteristics of myelin protein.J. Neurochem. 182119–2128.
Haley, J. E., and Ledeen, R. W. (1979). Incorporation of axonally transported substances into myelin lipids.J. Neurochem. 32735–742.
Karlsson, J. -O., and Sjostrand, J. (1971). Synthesis, migration and turnover of protein in retinal ganglion cells.J. Neurochem. 18749–767.
Laatsch, R. H., Kies, M. M., Gordon, S., and Alvord, E. C., Jr. (1962). The encephalomyelitic activity of myelin isolated by ultracentrifugation.J. Exp. Med. 115777–788.
Ledeen, R. W., and Yu, R. K. (1978). Methods for isolation and analysis of gangliosides. InResearch Methods in Neurochemistry (Marks, N., and Rodnight, R., Ed.), Plenum Press, New York, pp. 371–410.
Ledeen, R. W., Yu, R. K., and Eng, L. F. (1973). Gangliosides of human myelin: Sialosylgalactosylceramide (G7) as a major component.J. Neurochem. 21829–839.
Ledeen, R. W., Skrivanek, J. A., Tirri, L. J., Margolis, R. K., and Margolis, R. U. (1976). Gangliosides of the neuron: localization and origin. InGanglioside Function: Biochemical and Pharmacological Implications (Porcellati, G., Ceccarelli, B., and Tettamanti, G., Ed.), Plenum Press, New York, pp. 83–103.
Ledeen, R. W., Cochran, F. B., Yu, R. K., Samuels, F. G., and Haley, J. E. (1980). Gangliosides of the CNS myelin membrane. InStructure and Function of the Gangliosides (Svennerholm, L., Mandel, P., Dreyfus, H., and Urban, P. -F., Eds), Plenum Press, New York, pp. 167–176.
Lindberg, O., and Ernster, L. (1956). Determination of organic phosphorus compounds by phosphate analysis. InMethods of Biochemical Analysis (Gluck, D., Ed.), Interscience, New York, pp. 7–9.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193265–275.
Maizel, J. V., Jr. (1966). Acrylamide-gel electrophorograms by mechanical fractionation: Radioactive adenovirus proteins.Science 151988–990.
Marinetti, G. V., Erbland, J., and Stotz, E. (1959). The quantitative analysis of plasmalogens by paper chromatography.Biochim. Biophys. Acta 31251–252.
McEwen, B. S., and Grafstein, B. (1968). Fast and slow components in axonal transport of protein.J. Cell Biol. 38494–508.
Morell, P., Lipkind, R., and Greenfield, S. (1973). Protein composition of myelin from brain and spinal cord of several species.Brain Res. 58510–514.
Norton, W. T. (1977). Isolation and characterization of myelin. InMyelin (Morell, P., Ed.), Plenum Press, New York, pp. 161–199.
Norton, W. T., and Autilio, L. A. (1966). The lipid composition of purified bovine brain myelin.J. Neurochem. 13213–222.
Norton, W. T., and Poduslo, S. (1973). Myelination in rat brain: Method of myelin isolation.J. Neurochem. 21749–757.
Peters, A., Palay, S. L., and Webster, H. DeF. (1976).The Fine Structure of the Nervous System: The Neurons and Supporting Cells, W. B. Saunders, Philadelphia, pp. 191–226.
Prensky, A. L., Fujimoto, K., and Agrawal, H. C. (1975). Are myelin proteins synthesized in retinal ganglion cells?J. Neurochem. 25883–887.
Prohaska, J. R., Clark, D. A., and Wells, W. W. (1973). Improved rapidity and precision in the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase.Anal. Biochem. 56275–282.
Samuels, F. G., Haley, J. E., and Ledeen, R. W. (1979). Purity of myelin isolated in presence of EGTA. Abstracts, American Society for Neurochemistry, Tenth Annual Meeting, 89.
Schlaepfer, W. W., and Bunge, R. P. (1973). Effects of calcium ion concentration on the degeneration of amputated axons in tissue culture.J. Cell Biol. 59456–470.
Suzuki, K. (1970). Formation and turnover of myelin ganglioside.J. Neurochem. 17209–213.
Suzuki, K., Poduslo, S. E., and Norton, W. T. (1967). Gangliosides in the myelin fraction of developing rats.Biochim. Biophys. Acta 144375–381.
Suzuki, K., Poduslo, J. F., and Poduslo, S. E. (1968). Further evidence for a specific ganglioside fraction closely associated with myelin.Biochim. Biophys. Acta 152576–586.
Svennerholm, L. (1957). Quantitative estimation of sialic acids. II. Colorimetric resorcinol-hydrochloric acid method.Biochim. Biophys. Acta 24604–611.
Svennerholm, L. (1963). Chromatographic separation of human brain gangliosides.J. Neurochem. 10613–623.
Ueno, K., Ando, S., and Yu, R. K. (1978). Gangliosides of human, cat, and rabbit spinal cords and cord myelin.J. Lipid Res. 19863–871.
Waxman, S. G., and Foster, R. E. (1980). Ionic channel distribution and heterogeneity of the axon membrane in myelinated fibers.Brain Res. Rev. 2205–234.
Willard, M., Cowan, W. M., and Vagelos, P. R. (1974). The polypeptide composition of intra-axonally transported proteins: Evidence for four transport velocities.Proc. Nat. Acad. Sci. USA 712183–2187.
Yu, R. C. -P., and Bunge, R. P. (1975). Alterations in the peripheral myelin sheath and node of Ranvier produced by treatment with trypsin.J. Cell Biol. 641–14.
Yu, R. K., and Ledeen, R. W. (1970). Gas-liquid chromatographic assay of lipid-bound sialic acids: Measurement of gangliosides in brain of several species.J. Lipid Res. 11506–516.
Yu, R. K., and Yen, S. I. (1975). Gangliosides in developing mouse brain myelin.J. Neurochem. 25229–232.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Haley, J.E., Samuels, F.G. & Ledeen, R.W. Study of myelin purity in relation to axonal contaminants. Cell Mol Neurobiol 1, 175–187 (1981). https://doi.org/10.1007/BF00710718
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00710718