Ross S M, Sabri M I, Spencer P S
J Neurochem. 1983 Jul;41(1):222-9. doi: 10.1111/j.1471-4159.1983.tb11831.x.
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.
已从富含89%静止雪旺细胞的外周神经中获得了富含质膜的组分。这些组分是从猫坐骨神经在大腿上部横断和缝合8 - 10周后去鞘远端残端的束内组织制备的。将富含雪旺细胞的组织切碎、匀浆并离心以去除细胞核和未分散的组织。对所得上清液进行离心产生沉淀，对沉淀进行渗透压休克处理，铺在不连续蔗糖梯度上，然后再次离心。将富含质膜（PM）标志物的组分合并，进行16小时的渗透压休克处理，铺在第二个不连续蔗糖密度梯度上，然后再次离心。膜组分（0.6M:0.85M和0.85M:1.0M界面）含有均匀的单层囊泡群体，无髓磷脂。0.85M组分中5'-核苷酸酶、2',3'-环核苷酸3'-磷酸水解酶和特异性[3H]哇巴因结合分别比粗匀浆富集4.8倍、3.0倍和5.7倍。这些组分中琥珀酸脱氢酶、乳酸脱氢酶和葡萄糖-6-磷酸酶的酶活性也较低（分别为对照值的9%、13%和15%）。质膜组分（0.85M）的蛋白质产量约为0.6mg/g失神经神经。该制剂应适合于表征不受神经元调节的雪旺细胞的表面特性。
