THE CELLULAR ORGANIZATION OF CHOLESTEROL BIOSYNTHESIS

胆固醇生物合成的细胞组织

• 批准号: 3343794
• 负责人: YVONNE LANGE
• 金额: $ 14.03万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3343794
• 关键词: Golgi apparatus; antibody specificity; blood lipoprotein metabolism; blood lipoprotein transport; cholesterol; density gradient ultracentrifugation; endoplasmic reticulum; enzyme mechanism; fibroblasts; glycoproteins; high performance liquid chromatography; immunological substance; intracellular; intracellular membranes; lipid biosynthesis; low density lipoprotein; lysosomes; membrane permeability; membrane proteins; phospholipids; pinocytosis; radionuclides; radiotracer; receptor mediated endocytosis; steroid biosynthesis; thin layer chromatography; tissue /cell culture; tritium

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.

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。 起点是我们最近在有文化的人类中的演示 成纤维细胞中几种甾醇中间体不均匀 分布在亚细胞部分中。 这导致了 胆固醇生物合成尚未完成的假设 位于平滑内质网的单个位点。 这 所提出的方法将涉及细胞的亚细胞分离 通过平衡蔗糖密度离心匀浆，速率 蔗糖梯度和两相水相中的分区沉降 分割。 培养的成纤维细胞将与 3H-乙酸盐一起孵育 生物合成标记以诱导积累 中间体角鲨烯、氧化角鲨烯和羊毛甾醇：(a) 10℃孵育； (b)用4、4、10β治疗- Trimethyl-trans-decal-3 beta-ol，2,3-氧化角鲨烯抑制剂 环化酶：和（c）暴露于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 其前体将与标记的分布进行比较 对于光滑和粗糙内质网、高尔基体、 细胞核和质膜。 由于洋地黄皂苷形成特定的， 膜中与胆固醇的化学计量复合物，从而 增加它们的密度，它将被用来区分什么 点前体甾醇与富含胆固醇相关 膜。 我们建议采用洋地黄皂苷作为探针 富含胆固醇和羊毛甾醇的鉴定和分离 通过制备毛地黄皂苷抗体并制备 与同源物反应的洋地黄皂苷的半乳糖基化衍生物 凝集素。 另外两个具体假设也将得到检验：(1) 酸性囊泡携带细胞内胆固醇前体？ (2) 是新合成物质的传递途径 胆固醇对质膜的影响与新的相关 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拓扑结构和 导致细胞内胆固醇浓度的机制 表面膜具有根本意义。