Isolation and partial characterization of plasmalemma from quiescent Schwann cells in denervated cat sciatic nerve.
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.
D015720:5'-Nucleotidase; D000818:Animals; D002415:Cats; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D003714:Denervation; D005260:Female; D008297:Male; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000032:analysis; D009708:Nucleotidases / Q000032:analysis; D012583:Schwann Cells / Q000648:ultrastructure*; D012584:Sciatic Nerve / Q000648:ultrastructure*
D009419:Nerve Tissue Proteins; D009708:Nucleotidases; D015720:5'-Nucleotidase