Abstract
Myelinated axons were isolated by flotation from bovine pons, middle cerebellar peduncle, cervical spinal cord and three regions of the subcortical white matter. The myelinated axons were osmotically and mechanically shocked, followed by fractionation on a linear 15% sucrose to 45% sucrose density gradient. Axolemma-enriched fractions (AEF) found in the 28% to 32% sucrose region of the gradient from brainstem and cord white matter had high acetylcholinesterase (AChE) while little or nil AChE activity was found in corresponding AEF derived from the subcortical white matter. Morphologically, the subcortical white matter from all regions contained a heterogeneous population of well-myelinated to thinly myelinated axons, while brainstem and cord regions contained a more homogeneous population of well-myelinated axons. Histochemical analysis of AChE localized this enzyme to axonal elements. The AEF derived from any white matter source had similar polypeptide compositions. AEF derived from subcortical white matter contained two-fold more myelin basic protein and a three-fold greater content of 2′ 3′ cyclic nucleotide 3′ phosphodiesterase (CNP) compared with AEF derived from well myelinated white matter. We conclude that the purity of the AEF is related to the degree of myelination of the white matter from which the AEF is derived. Homogeneously well myelinated white matter (pons, cerebellar peduncle, cervical spinal cord) yields the highest purity AEF, as judged by the low CNP and myelin basic protein content and highest enrichment in AChE specific activity.
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DeVries, G. H. 1984. The axonal plasma membrane. Pages 341–360,in Lajtha, A. (ed.) Handbook of Neurochemistry, Plenum Press, New York.
DeVries, G. H. 1981. Isolation of axolemma-enriched fractions from mammalian CNS. Pages 3–38,in Marks, N., and Rodnight, R. (eds.) Research Methods in Neurochemistry, Vol. 5, Plenum Press, New York.
DeVries, G. H., Payne, W., and Saul, R. 1981. Composition of axolemma-enriched fractions isolated from bovine CNS myelinated axons. Neurochem. Res. 6:521–537.
DeVries, G. H., Anderson, M. G., and Johnson, D. 1983. Fractionation of isolated CNS myelinated axons via sucrose density centrifugation in a zonal rotor. J. Neurochem. 40:1709–1717.
Yoshino, J., Griffin, J., and DeVries, G. H. 1983. Identification of an axolemma-enriched fraction from peripheral nerve. J. Neurochem. 41:1126–1130.
Hall, Z. W. 1972. Multiple forms of acetylcholinesterase and their distribution in end plate and non-end plate regions of rat diaphragm muscle. J. Neurobiol. 4:343–361.
Sogin, D. C. 1976. 2′3′ cyclic NADP as a substrate for 2′3′ cyclic nucleotide 3′ Phosphohydrolase. J. Neurochem. 27:1333–1337.
Barnett, R. E. 1970. Effect of monovalent cations on the ouabain inhibition of sodium and potassium ion activated adenosine triphosphate. Biochemistry 9:4644–4648.
Hartree, E. F. 1972. Determination of protein: A modification of the lowry method that gives a linear photometric response. Anal. Biochem. 48:422–427.
El-Badawi, A., and Schenk, E. A. 1967. Histochemical methods for separate, consecutive and simutlaneous demonstration of acetylcholinesterase and norepinephrine in cyrostat sections. J. Histochem. Cytochem. 15:580–588.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacterophage T-4. Nature, 227:680–685.
Calabrese, V. P., Wallen, W., Castellano, G., Ward, L., Anderson, W. G., and DeVries, G. H. 1981. Enzyme-linked immunosorbent assay (ELISA) for antibodies to human myelin and axolemma-enriched fractions. Neurosci. Lett. 21:189–195.
Philips, J. M., and Sweadner, K. J., 1986. Nodal and paranodal distribution of isozymes of Na, K-ATPase. Soc. Neurosci. Abst. 12:161.
DeVries, G. H. 1976. Isolation of axolemma-enriched fractions from bovine central nervous system. Neurosci. Lett. 3:117–122.
DeVries, G. H., Norton, W. T., and Raine, C. S. 1972. Axons: isolation from mammalian central nervous system. Science, 175:1370–1372.
Zetusky, W., Calabrese, V. P., Zetusky, A., Anderson, M. G., Cullen, M., and DeVries, G. H. 1979. Isolation and partial characterization of human CNS axolemma-enriched fractions. J. Neurochem. 32:1103–1109.
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. 147:339–352.
Stanley, J., Saul, R. G., Hadfield, M. G., and DeVries, G. H. 1979. Mammalian CNS Axolemma: Histochemical evidence for axonal plasma membrane origin of bovine and rat axolemma-enriched fractions. Neuroscience 4:155–167.
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Detskey, P.Z., Bigbee, J.W. & DeVries, G.H. Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS. Neurochem Res 13, 449–454 (1988). https://doi.org/10.1007/BF01268880
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DOI: https://doi.org/10.1007/BF01268880