Lange Y, Steck T L
J Biol Chem. 1985 Dec 15;260(29):15592-7.
The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.
本研究检测了新合成的甾醇在培养的人成纤维细胞中的分布情况。我们首先证明，胆固醇总量和外源添加的[3H]胆固醇都是质膜的标志物，可能比5'-核苷酸酶更好。将细胞与放射性乙酸孵育以生物合成标记其内源性甾醇，用胆固醇氧化酶处理以将质膜胆固醇转化为胆甾烯酮，然后匀浆并在蔗糖梯度上离心至平衡。使用这些标志物监测各种细胞器的密度梯度分布：质膜，放射性胆甾烯酮；滑面内质网，3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶）；以及高尔基体，半乳糖基转移酶。放射性细胞内胆固醇和羊毛甾醇的浮力密度分布在1.12 g/cm3处都有一个峰值，类似于5'-核苷酸酶和半乳糖基转移酶，但与HMG-CoA还原酶不同。这一结果表明，胆固醇生物合成在内质网中并未完成。洋地黄皂苷处理使质膜和细胞内胆固醇的分布向更高密度移动。用胆固醇氧化酶对完整细胞进行预处理消除了质膜的洋地黄皂苷移动，但没有消除细胞内胆固醇的移动，表明这两个膜池并非完全物理相关。由于细胞内胆固醇的移动比任何细胞器标志物都要大，它一定存在于一个单独的膜中。由于洋地黄皂苷选择性地改变富含胆固醇的膜的密度，我们推断新合成的胆固醇在被输送到质膜之前会在这种膜中积累。综上所述，这些结果表明，胆固醇可能通过在离散但未明确的细胞内膜中由甾醇前体如羊毛甾醇合成而被浓缩，以便输送到质膜。
