Analysis of the distribution of cholesterol in the intact cell.
Analysis of the distribution of cholesterol in the intact cell.
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DOI：
10.1016/s0021-9258(17)43782-3
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发表时间：
1983-12
期刊：
影响因子：
--
通讯作者：
Y. Lange;B. Ramos
中科院分区：
文献类型：
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作者：
Y. Lange;B. Ramos
我们已经使用的酶胆固醇氧化酶，催化胆固醇的氧化胆甾-4-烯-3-酮，研究胆固醇在培养的成纤维细胞，中国仓鼠卵巢细胞，和分离的大鼠肝细胞的分布。虽然质膜通常不受胆固醇氧化酶的攻击，但我们发现用低离子强度缓冲液和戊二醛处理细胞使其胆固醇极易氧化。大部分胆固醇在所有三种细胞类型中被氧化：成纤维细胞中为94%，中国仓鼠卵巢细胞中为92%，肝细胞中为80%。考虑到酶只能到达细胞的外表面，并且胆固醇可以快速穿过固定的质膜，这些值反映了质膜中存在的细胞胆固醇的分数。进一步的实验证实了这一解释。通过短暂暴露于外源性放射性标记，用[3 H]胆固醇标记成纤维细胞，并与[14 C]甲羟戊酸孵育以生物合成标记胆固醇。胆固醇氧化酶攻击至少97%的外源标记，但只有10%的生物合成标记的胆固醇。这些数据表明，胆固醇氧化酶没有达到细胞内池和细胞膜中的胆固醇不是在快速平衡与内膜。胆固醇从生物合成标记[3 H]胆固醇和外源标记[14 C]胆固醇的细胞转移到血浆的研究证实了两种标记物的不同亚细胞分布。这些研究表明，出乎意料的高比例的细胞胆固醇与质膜相关，并且这种胆固醇池可以快速和选择性地标记和氧化。这些特征使得胆固醇成为质膜的有用的特异性标记物。
We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.