CELLULAR MEMBRANES - ISOLATION AND CHARACTERIZATION OF PLASMA AND SMOOTH MEMBRANES OF HELA CELLS
CELLULAR MEMBRANES - ISOLATION AND CHARACTERIZATION OF PLASMA AND SMOOTH MEMBRANES OF HELA CELLS
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DOI：
10.1016/0003-9861(68)90008-8
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发表时间：
1968-01-01
影响因子：
3.9
通讯作者：
EYLAR, EH
中科院分区：
文献类型：
--
作者：
BOSMANN, HB;HAGOPIAN, A;EYLAR, EH
描述了一种使用不连续蔗糖梯度（20-45% 蔗糖）从 HeLa 细胞 Dounce 匀浆中获得的 4000g 上清液制备膜组分的方法。获得了两个部分，质膜和光滑内膜，通过电子显微镜观察，它们不含细胞核、线粒体、溶酶体、微粒体、核糖体、致密体或其他可识别的细胞元件。电子显微镜检查显示，质膜的完整性得以保留；通过相差显微镜观察到破裂的细胞包膜。质膜的纯度通过 5' 核苷酸酶的比活性比细胞匀浆高出 120 倍来表示。质膜中还发现了 ATP 酶、碱性磷酸酶和磷酸二酯酶，分别进行了 30 倍、21 倍和 6 倍的纯化。与质膜牢固结合的 5' 核苷酸酶仅位于这些膜中。基于该酶回收率的质膜产率为 40-50%。由于未能分别检测琥珀酸脱氢酶、酯酶和 UDP 酶，表明质膜部分不存在线粒体、微粒体和光滑膜。质膜的特点是胆固醇/磷脂摩尔比较高 (1.05)，与髓磷脂和红细胞基质中的摩尔比相似。光滑膜中的这一比率 (0.06) 说明了这些膜与质膜之间的组成存在巨大差异。与在 45% 蔗糖含量以下发现为颗粒的质膜相反，在 25% 蔗糖含量以上发现光滑的内膜。它们含有61%的脂质；质膜只有 29% 的脂质。两部分都含有大量的 RNA，但都含有胆固醇、己糖胺和唾液酸。光滑的内膜具有最高的UDPase（一种高尔基酶）含量和比活性。但未检测到 5' 核苷酸酶和 ATP 酶、质膜酶以及琥珀酸脱氢酶（一种线粒体酶）。光滑膜中发现的其他酶包括碱性磷酸酶、酯酶和磷酸二酯酶。两种膜组分中所含蛋白质的氨基酸组成相似；光滑内膜蛋白质的特征是每1000个氨基酸残基有151个碱性氨基酸残基和221个酸性残基，而质膜蛋白质每1000个氨基酸残基含有193个碱性氨基酸残基和188个酸性残基。两个级分中的谷氨酸含量均超过其他氨基酸。
A method is described for preparation of membrane fractions from the 4000gsupernatant fluid, obtained from a Dounce homogenate of HeLa cells, using a discontinuous sucrose gradient (20–45% sucrose). Two fractions were obtained, the plasma membranes and the smooth internal membranes that appeared by electron microscopy to be free of nuclei, mitochondria, lysosomes, microsomes, ribosomes, dense bodies, or other recognizable cellular elements. The integrity of the plasma membranes were preserved as shown by electron microscopic examination; ruptured cellular envelopes were seen by phase microscopy. The degree of purity of the plasma membranes was shown by high increase in specific activity of the 5′ nucleotidase over the cellular homogenate of 120 fold. An ATPase, alkaline phosphatase, and phosphodiesterase were also found in the plasma membranes, giving a 30-fold, 21-fold, and 6-fold purification respectively. The 5′ nucleotidase, strongly bound to the plasma membranes, was located exclusively in these membranes. The yield of plasma membranes based on recovery of this enzyme was 40–50%. The absence of mitochondria, microsomes, and smooth membranes from the plasma membrane fraction was revealed by the failure to detect succinic dehydrogenase, esterase, and UDPase, respectively. The plasma membranes were characterized by a high molar ratio of cholesterol/phospholipid (1.05) which is similar to that found in myelin, and erythrocyte stroma. This ratio in the smooth membranes (0.06) illustrates the large difference in composition between these membranes and the plasma membranes. In contrast to the plasma membranes, found as a pellet below the 45% sucrose, the smooth internal membranes were found above the 25% sucrose level. They contained 61% lipid; the plasma membranes had only 29% lipid. Neither fraction contained appreciable amounts of RNA, but both had cholesterol, hexosamine, and sialic acid. The smooth internal membranes had the highest content and specific activity of UDPase, a Golgi enzyme. but the 5′ nucleotidase and ATPase, the plasma membrane enzymes, and succinic dehydrogenase, a mitochondrial enzyme, were not detected. Other enzymes found in the smooth membranes included alkaline phosphatase, esterase, and phosphodiesterase. The amino acid composition of the protein contained in the two membrane fractions was similar; the protein of the smooth internal membranes was characterized by 151 basic amino acid residues per 1000 residues and 221 acidic residues while the plasma membrane protein contained 193 basic and 188 acidic residues per 1000 amino acid residues. The glutamic acid content exceeded the other amino acids in both fractions.