Lange Y, Muraski M F
Department of Pathology, Rush Medical College, Chicago, Illinois 60612-3864.
J Biol Chem. 1988 Jul 5;263(19):9366-73.
We have examined the membrane topography of cholesterol biosynthesis in cultured human fibroblasts. We fed the cells with radioacetate and then interrupted the biosynthetic pathway so as to trap labeled intermediates in their subcellular locations. We analyzed homogenates of human fibroblasts labeled biosynthetically from radioacetate by centrifugation to equilibrium on sucrose gradients. The following two methods were used to interrupt cholesterol biosynthesis: incubation at 10 degrees C and treatment with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase. Incubation at 10 degrees C caused the accumulation of radiolanosterol at the expense of cholesterol. The lanosterol appeared predominantly at an unusually buoyant density (20% (w/w) sucrose; d = 1.08 g/cm3) as well as at the density normally labeled at 37 degrees C (30% sucrose; d = 1.13 g/cm3). 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol treatment caused the accumulation of labeled squalene and squalene 2,3-oxide. Reversal of the block permitted the label to progress rapidly as a wave into lanosterol and ultimately into cholesterol. The profiles of the three precursors did not coincide, suggesting that they were mostly in different membranes. Squalene was uniquely confined to a density of 1.18 g/cm3 (40% sucrose) while squalene 2,3-oxide appeared in peaks of density 1.08 g/cm3 and 1.13 g/cm3 (20% and 30% sucrose). Lanosterol was in a peak of density 1.13 g/cm3. Pulse-chase experiments showed that lanosterol synthesized in the membranes at 20% sucrose moved rapidly to the membranes at 30% sucrose where it was converted to cholesterol. The density gradient profiles of the following organelle markers also were monitored: plasma membrane, cholesterol mass; Golgi apparatus, galactosyltransferase; endoplasmic reticulum, RNA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cytochrome c reductase; peroxisomes, catalase. None of these markers appeared at the buoyant density of 1.08 g/cm3. We conclude that 1) cholesterol biosynthesis may be topographically heterogeneous and 2) newly synthesized squalene 2,3-oxide resides in a buoyant membrane fraction distinct from markers for the major organelles.
我们研究了培养的人成纤维细胞中胆固醇生物合成的膜拓扑结构。我们用放射性醋酸盐喂养细胞，然后中断生物合成途径，以便将标记的中间体捕获在其亚细胞位置。我们通过在蔗糖梯度上离心至平衡，分析了由放射性醋酸盐生物合成标记的人成纤维细胞匀浆。采用以下两种方法中断胆固醇生物合成：在10℃孵育和用4,4,10β-三甲基-反式-十氢萘-3β-醇处理，后者是氧化角鲨烯环化酶的特异性抑制剂。在10℃孵育导致放射性羊毛甾醇积累，胆固醇减少。羊毛甾醇主要出现在异常轻的密度（20%（w/w）蔗糖；d = 1.08 g/cm³）以及在37℃正常标记的密度（30%蔗糖；d = 1.13 g/cm³）。4,4,10β-三甲基-反式-十氢萘-3β-醇处理导致标记的角鲨烯和角鲨烯2,3-氧化物积累。阻断的逆转使标记物作为一个波迅速进展为羊毛甾醇并最终进展为胆固醇。这三种前体的分布曲线不一致，表明它们大多位于不同的膜中。角鲨烯独特地局限于密度为1.18 g/cm³（40%蔗糖），而角鲨烯2,3-氧化物出现在密度为1.08 g/cm³和1.13 g/cm³（20%和30%蔗糖）的峰中。羊毛甾醇处于密度为1.13 g/cm³的峰中。脉冲追踪实验表明，在20%蔗糖的膜中合成的羊毛甾醇迅速转移到30%蔗糖的膜中，并在那里转化为胆固醇。还监测了以下细胞器标志物的密度梯度分布：质膜，胆固醇总量；高尔基体，半乳糖基转移酶；内质网，RNA、3-羟基-3-甲基戊二酰辅酶A还原酶和细胞色素c还原酶；过氧化物酶体，过氧化氢酶。这些标志物均未出现在1.08 g/cm³的轻密度处。我们得出结论：1）胆固醇生物合成在拓扑结构上可能是异质的；2）新合成的角鲨烯2,3-氧化物存在于一个与主要细胞器标志物不同的轻膜组分中。
