Isolation and partial characterization of plasmalemma from quiescent Schwann cells in denervated cat sciatic nerve

S. M. Ross, M. I. Sabri, Peter Spencer

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Abstract

Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89% in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8-10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M: 0.85 M and 0.85 M:1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5'-nucleotidase, 2',3'-cyclic nucleotide 3'-phosphohydrolase, and specific [3H]ouabain binding, 4.8-, 3.0-, and 5.7-fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatase (9, 13, and 15% of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.

Original languageEnglish (US)
Pages (from-to)222-229
Number of pages8
JournalJournal of Neurochemistry
Volume41
Issue number1
StatePublished - 1983
Externally publishedYes

Fingerprint

Schwann Cells
Sciatic Nerve
Cell membranes
nucleotidase
Cats
Cells
Cell Membrane
Tissue
Sucrose
Glucose-6-Phosphatase
Unilamellar Liposomes
5'-Nucleotidase
Succinate Dehydrogenase
Centrifugation
Surface Properties
Cyclic Nucleotides
Enzyme activity
Ouabain
Myelin Sheath
Thigh

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Isolation and partial characterization of plasmalemma from quiescent Schwann cells in denervated cat sciatic nerve. / Ross, S. M.; Sabri, M. I.; Spencer, Peter.

In: Journal of Neurochemistry, Vol. 41, No. 1, 1983, p. 222-229.

Research output: Contribution to journalArticle

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abstract = "Fractions enriched in plasma membranes have been obtained from peripheral nerves enriched 89{\%} in quiescent Schwann cells. Fractions were prepared from the intrafascicular tissue of desheathed distal stumps of cat sciatic nerve 8-10 weeks after transection and suture in the upper thigh. Tissue enriched in Schwann cells was minced, homogenized, and centrifuged to remove nuclei and undispersed tissue. Centrifugation of the resulting supernatant produced a pellet that was osmotically shocked, layered over a discontinuous sucrose gradient, and recentrifuged. Fractions enriched in plasma membrane (PM) markers were pooled, osmotically shocked for 16 h, layered over a second discontinuous sucrose density gradient, and recentrifuged. Membrane fractions (0.6 M: 0.85 M and 0.85 M:1.0 M interfaces) contained a homogeneous population of unilamellar vesicles free of myelin. The 0.85 M fraction was enriched in 5'-nucleotidase, 2',3'-cyclic nucleotide 3'-phosphohydrolase, and specific [3H]ouabain binding, 4.8-, 3.0-, and 5.7-fold over the crude homogenate, respectively. These fractions also demonstrated low enzyme activities for succinate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphatase (9, 13, and 15{\%} of control values, respectively). Protein yield of the PM fraction (0.85 M) was approximately 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.",
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