THE CELLULAR ORGANIZATION OF CHOLESTEROL BIOSYNTHESIS

本研究的目的是阐明地形 哺乳动物细胞内胆固醇生物合成的组织。 出发点是我们最近在培养的人类 成纤维细胞认为几种固醇中间体的分布不均匀 分布在亚细胞组分中。 这导致了 胆固醇生物合成未完成的假设 位于滑面内质网的一个位点。 的 所提出的方法将涉及细胞的亚细胞分级分离 通过平衡蔗糖密度离心匀浆，速率 在蔗糖梯度和两相水溶液中的带式沉降 分区 将培养的成纤维细胞与3 H-乙酸盐一起孵育 以生物合成的方式标记，从而诱导 中间体角鲨烯、氧化角鲨烯和羊毛甾醇：（a） 在10 ℃下孵育;（B）用4，4，10 β- 2，3-氧化角鲨烯抑制剂三甲基-反式-癸烷-3 β-醇 环化酶：和（c）暴露于未鉴定的环化酶抑制剂， 我们发现羊毛甾醇转化为胆固醇 最近 新生胆固醇的亚细胞分布， 将其前体与标记物的分布进行比较 对于光滑和粗糙内质网，高尔基体， 细胞核和质膜。 由于毛地黄皂苷形成一种特异性的， 与膜中胆固醇的化学计量复合物， 增加了它们的密度，它将被用来区分 点前体固醇成为与胆固醇丰富 膜。 我们建议调整毛地黄皂苷作为探针， 富含胆固醇和羊毛甾醇化合物的鉴定和分离 通过制备毛地黄皂苷的抗体和制备 毛地黄皂苷的半乳糖基化衍生物 凝集素。 另外两个具体的假设也将被测试：（1） 酸性囊泡携带的细胞内胆固醇前体？ (2)是新合成的 胆固醇对细胞膜的作用与那些新的 合成磷脂和蛋白质 鉴于中央 胆固醇在人类心血管疾病中的作用 了解胆固醇生物合成的地形和 导致细胞中胆固醇浓度的机制 表面膜是基本的兴趣。

The goal of this research is the elucidation of the topographic organization of cholesterol biosynthesis within mammalian cells. The starting point is our recent demonstration in cultured human fibroblasts that several sterol intermediates are nonuniformly distributed among subcellular fractions. This has lead to the hypothesis that cholesterol biosynthesis is not taken to completion at a single locus in the smooth endoplasmic reticulum. The proposed approach will involve subcellular fractionation of cell homogenates by equilibrium sucrose density centrifugation, rate zonal sedimentation in sucrose gradients and two phase aqueous partition. Cultured fibroblasts will be incubated with 3H-acetate to label biosynthetically so as to induce the accumulation of the intermediates squalene, squalene oxide and lanosterol: (a) incubation at 10 degrees C; (b) treatment with 4,4,10 beta - Trimethyl-trans-decal-3 beta-ol, an inhibitor of 2,3-oxidosqualene cyclase: and (c) exposure to an unidentified inhibitor of the conversion of lanosterol to cholesterol which we discovered recently. The subcellular distribution of nascent cholesterol and its precursors will be compared with the distribution of markers for the smooth and rough endoplasmic reticulum, Golgi apparatus, nucleus and plasma membrane. Since digitonin forms a specific, stoichiometric complex with cholesterol in membranes and thereby increases their density, it will be used to distinguish at what point precursor sterols become associated with cholesterol-rich membranes. We propose to adapt digitonin as a probe for the identification and isolation of cholesterol- and lanosterol-rich membranes by making antibodies to digitonin and by preparing galactosylated derivatives of digitonin reactive with cognate lectins. Two other specific hypotheses also will be tested: (1) Are intracellular cholesterol precursors carried by acidic vesicles? (2) Is the pathway for the delivery of newly synthesized cholesterol to the plasma membrane related to those for newly synthesized phospholipids and proteins? In view of the central role of cholesterol in human cardiovascular disease, an understanding of the topography of cholesterol biosynthesis and the mechanism leading to the concentration of cholesterol in the cell surface membrane is of fundamental interest.