Isolation and Partial Characterization of Plasmalemma from Quiescent Schwann Cells in Denervated Cat Sciatic Nerve

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Abstract
Fractions enriched in plasma membranes were 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 wk 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 2nd 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 .apprx. 0.6 mg/g of denervated nerve. This preparation should be suitable to characterize the surface properties of Schwann cells free of neuronal regulation.