THE CELLULAR ORGANIZATION OF CHOLESTEROL BIOSYNTHESIS

这项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。 起点是我们最近在培养的人类身上的演示。 几种甾醇中间产物不均匀的成纤维细胞 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说 在光滑内质网中的一个单一位置。这个 建议的方法将涉及细胞的亚细胞分离 平衡蔗糖密度离心法匀浆 蔗糖梯度和两相水相中的带状沉积 分区。培养的成纤维细胞将与~3H-乙酸酯孵育 以生物合成的方式标记，以诱导 中间体角鲨烯、氧化角鲨烯和羊毛甾醇：(A) 10℃孵化；(B)用4，4，10β- 2，3-氧化桂烯的抑制剂三甲基-反式-3-β-醇 环化酶：和(C)暴露于一种不明的环化酶抑制剂 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和 它的前驱将与标记的分布进行比较 对于光滑和粗糙的内质网，高尔基体， 胞核和质膜。由于洋地黄素形成了一种特定的， 膜中胆固醇的化学计量比络合物，从而 增加它们的密度，它将被用来区分什么是 点状前体甾醇与富含胆固醇相关 膜。我们建议将洋地黄素改编为探针，用于 富含胆固醇和羊毛甾醇的化合物的鉴定与分离 通过制作洋地黄素抗体和制备 洋地黄素半乳糖基衍生物与同源化合物的反应 凝集素。 另外两个具体的假设也将得到检验：(1) 酸性小泡携带的细胞内胆固醇前体？ (2)是新合成药物的传递途径 胆固醇对质膜的影响与新近形成的 人工合成的磷脂和蛋白质？鉴于中环 胆固醇在人类心血管疾病中的作用 对胆固醇生物合成的拓扑学的理解 导致细胞内胆固醇浓度升高的机制 表面膜是最重要的。

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.