Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts.
Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts.
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发表时间：
1989-03
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通讯作者：
Y. Lange;M. Swaisgood;B. Ramos;T. Steck
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作者：
Y. Lange;M. Swaisgood;B. Ramos;T. Steck
文献表明，胆固醇和鞘磷脂可能基本上局限于哺乳动物细胞的质膜;然而，这一前提迄今尚未得到直接检验。我们从三个方面探讨了这个问题。首先，我们通过平衡蔗糖密度梯度离心分离培养的人成纤维细胞的全匀浆。我们发现，胆固醇和鞘磷脂的配置文件是无法区分的两个质膜标记，5'核苷酸酶和[3 H]半乳糖，这是共轭的外源供体的完整细胞的表面通过半乳糖基转移酶。其次，我们确定了完整细胞的相对表面积，从他们的摄取1-（4-三甲基氨基）苯基-6-苯基己-1，3，5-三烯，阳离子荧光染料，分区，但不跨越质膜。相对于人红细胞血影，成纤维细胞的表观表面积为17，500 m2/细胞，而犬肝细胞的表观表面积为11，500 m2/细胞。细胞胆固醇与染料结合（因此，表面积）的相对比率在血影、成纤维细胞和肝细胞中非常相似，分别为1.0、1.12和0.67。最后，我们发现胆固醇和鞘磷脂与5'核苷酸酶的特定比率在梯度纯化的质膜中仅比在整个匀浆中低10%。使用完全不同的纯化方法获得了类似的结果：两相水分配。胆固醇和鞘磷脂的馏分中富含其他膜是密切成比例的5'核苷酸酶的含量，这表明这些脂质的存在反映了质膜碎片的污染。纯化的质膜部分中的5'核苷酸酶/磷脂比大约是全细胞中的两倍。我们的结论是，在培养的人成纤维细胞中，5'核苷酸酶标记的隔室含有约90%的两种命名的脂质和一半的细胞磷脂磷。
The literature suggests that cholesterol and sphingomyelin might be essentially confined to plasma membranes in mammalian cells; however, this premise has thus far escaped a direct test. We explored the issue in three ways. First, we fractionated whole homogenates of cultured human fibroblasts by equilibrium sucrose density gradient centrifugation. We found that the profiles of cholesterol and sphingomyelin were indistinguishable from those of two plasma membrane markers, 5' nucleotidase and [3H]galactose, which was conjugated to the surface of intact cells from an exogenous donor by galactosyltransferase. Second, we determined the relative surface areas of intact cells from their uptake of 1-(4-trimethyl-amino)phenyl-6-phenylhexa-1,3,5-triene, a cationic fluorescent dye which partitions into but does not cross plasma membranes. Relative to human red cell ghosts, the apparent surface area of the fibroblasts was 17,500 microns2/cell while for canine hepatocytes, the value was 11,500 microns2/cell. The relative ratios of cell cholesterol to dye binding (hence, surface area) were quite similar in ghosts, fibroblasts, and liver cells; namely 1.0, 1.12, and 0.67, respectively. Finally, we found that the specific ratios of both cholesterol and sphingomyelin to 5' nucleotidase were only 10% less in gradient-purified plasma membranes than in whole homogenates. Similar results were obtained using an entirely different method of purification: two-phase aqueous partition. The cholesterol and sphingomyelin in fractions rich in other membranes was closely proportional to their 5' nucleotidase content, suggesting that the presence of these lipids reflected contamination by plasma membrane fragments. The 5' nucleotidase/phospholipid ratio in the purified plasma membrane fraction was roughly twice that in whole cells. We conclude that the compartment marked by 5' nucleotidase in cultured human fibroblasts contains approximately 90% of the two named lipids and half the cell phospholipid phosphorus.