ISOLATION OF PLASMA-MEMBRANES FROM HUMAN-SKIN FIBROBLASTS
ISOLATION OF PLASMA-MEMBRANES FROM HUMAN-SKIN FIBROBLASTS
复制标题
DOI：
10.1007/bf01868151
复制
发表时间：
1977-01-01
影响因子：
2.4
通讯作者：
RIORDAN, JR
中科院分区：
文献类型：
--
作者：
KARTNER, N;ALON, N;RIORDAN, JR
通过避免用蛋白酶或重金属处理的方法，从培养的人类皮肤成纤维细胞中大规模分离质膜。从滚瓶中收获细胞（0.5-1.0×109）并通过在低渗培养基中手动搅拌来破碎。然后通过常规差速离心和密度梯度离心获得富含质膜的级分（10-15mg蛋白质）。通过相差显微镜和电镜监测分级分离，测量标记酶的活性以及破碎前与细胞表面结合的 125I 麦芽凝集素的回收率。获得的膜主要是囊状的(直径0.1-2μm)并且在蔗糖密度为1.12时呈带状。此外，5''-核苷酸酶相对于细胞匀浆纯化了约20倍，并且在质膜富集级分中回收了近50%的该酶。 Mg2+-ATP酶、(Na++K+)Mg2+-ATP酶和前列腺素E以及氟化物刺激的腺苷酸环化酶的活性在该级分中也最大。在这些膜中，与细胞表面结合的 125I 麦芽凝集素的回收率和比活性最大。电子显微照片中唯一可识别的污染结构类似于高尔基体的平坦池板。唾液酸和半乳糖基转移酶活性的检测与这些结构的存在一致。制剂中各种标记物的进一步细分是可能的。类似的质膜富集级分也通过第二种方法获得，其中通过从滚瓶刮取细胞并通过在等渗介质中均质化来破碎来收获细胞。
Plasma membranes were isolated on a large scale from cultured human skin fibroblasts by methods which avoid treatment with proteases or heavy metals. Cells (0.5-1.0 .times. 109) were harvested from roller bottles and disrupted by manual agitation in hypotonic medium. A plasma membrane-enriched fraction (10-15 mg protein) was then obtained by conventional differential and density gradient centrifugation. Fractionation was monitored by phase-contrast microscopy and EM, measurement of activities of marker enzymes and recovery of 125I wheat germ agglutinin which was bound to the cell furface prior to disruption. The membranes obtained were mainly vesicular (0.1-2 .mu.m diameter) and banded at a sucrose density of 1.12. In addition, 5''-nucleotidase was purified approximately 20-fold with respect to the cell homogenate and nearly 50% of this enzyme was recovered in the plasma membrane-enriched fraction. Activities of Mg2+-ATPase, (Na+ + K+) Mg2+-ATPase and prostaglandin E, and fluoride-stimulated adenylate cyclases were also maximal in this fraction. Recovery and specific activity of 125I wheat germ agglutinin bound to the cell surface was greatest in these membranes. The only contaminating structures identifiable in electron micrographs resembled the flat cisternal plates of the Golgi apparatus. The detection of sialyl and galactosyl transferase activities was consistent with the presence of these structures. Some further subfractionation of the various markers in the preparation was possible. A similar plasma membrane-enriched fraction also was obtained by a 2nd method in which cells were harvested by scraping from the roller bottles and disrupted by homogenization in isotonic media.