Lange Y, Muraski M F
J Biol Chem. 1987 Apr 5;262(10):4433-6.
We have shown previously that newly synthesized lanosterol and cholesterol in homogenates of cultured human fibroblasts do not have the same equilibrium buoyant density as the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in the smooth endoplasmic reticulum (SER) (Lange, Y., and Steck, T. L. (1985) J. Biol. Chem. 260, 15592-15597). This finding suggested two alternative and novel hypotheses: (a) that lanosterol and cholesterol might be transported rapidly from the SER to other internal membranes or (b) that synthesis of the sterols is not associated with the SER, or at least not with that portion of this organelle bearing HMG-CoA reductase. We therefore compared the subcellular distribution of HMG-CoA reductase with that of enzymes which convert lanosterol to cholesterol. The two activities studied were the consumption of exogenous [3H]lanosterol and the conversion of exogenous radiolanosterol to radiocholesterol. Differential centrifugation, rate zonal centrifugation, and equilibrium sucrose gradient centrifugation of rat liver homogenates all showed that these enzyme activities did not comigrate with HMG-CoA reductase. The subcellular distribution of newly synthesized sterols also was examined in cultured human fibroblasts. Cells were incubated with radioactive acetate to label endogenous sterols biosynthetically, homogenized, and spun to equilibrium on sucrose gradients. The buoyant density profiles of radioactive cholesterol and lanosterol both had a peak at 1.12 g/cm3. Digitonin treatment shifted both sterols to higher densities, strong evidence that they resided in cholesterol-rich membranes. Pretreatment of intact cells with cholesterol oxidase, which selectively oxidizes plasma membrane cholesterol, abolished the digitonin shift of lanosterol but not of intracellular cholesterol. These findings provide support for the hypothesis that newly synthesized cholesterol and lanosterol are not in the same membrane.
我们之前已经表明，在培养的人成纤维细胞匀浆中，新合成的羊毛甾醇和胆固醇与滑面内质网（SER）中的3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶）具有不同的平衡浮力密度（兰格，Y.，和斯特克，T. L.（1985年）《生物化学杂志》260，15592 - 15597）。这一发现提出了两个替代的新假设：（a）羊毛甾醇和胆固醇可能从SER快速转运到其他内膜，或者（b）甾醇的合成与SER无关，或者至少与该细胞器中带有HMG-CoA还原酶的部分无关。因此，我们比较了HMG-CoA还原酶与将羊毛甾醇转化为胆固醇的酶的亚细胞分布。所研究的两种活性是外源性[3H]羊毛甾醇的消耗以及外源性放射性羊毛甾醇向放射性胆固醇的转化。对大鼠肝脏匀浆进行差速离心、速率区带离心和平衡蔗糖梯度离心均表明，这些酶活性与HMG-CoA还原酶不共迁移。还在培养的人成纤维细胞中检测了新合成甾醇的亚细胞分布。将细胞与放射性乙酸孵育以生物合成标记内源性甾醇，匀浆后在蔗糖梯度上离心至平衡。放射性胆固醇和羊毛甾醇的浮力密度分布在1.12 g/cm3处均有一个峰值。洋地黄皂苷处理使两种甾醇的密度都升高，这有力地证明它们存在于富含胆固醇的膜中。用胆固醇氧化酶对完整细胞进行预处理，该酶选择性地氧化质膜胆固醇，消除了羊毛甾醇的洋地黄皂苷密度变化，但未消除细胞内胆固醇的变化。这些发现为新合成的胆固醇和羊毛甾醇不在同一膜中的假设提供了支持。
