SQUALENE EPOXIDASE AND OXIDOSQUALENE CYCLASE FROM LIVER

来自肝脏的角鲨烯环氧化酶和氧化角鲨烯环化酶

• 批准号: 2182791
• 负责人: GLENN DOWNES PRESTWICH
• 金额: $ 13.39万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-03-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182791
• 关键词: SDS polyacrylamide gel electrophoresis; active sites; affinity labeling; clone cells; enzyme activity; enzyme inhibitors; enzyme substrate; farnesyl compound; genetic library; high performance liquid chromatography; human tissue; isomerase; laboratory rabbit; laboratory rat; lanosterol; liver; molecular cloning; oxygenases; polymerase chain reaction; protein purification; protein sequence; radiotracer; squalene

Squalene epoxidase (SE) and oxidosqualene cyclase (OSC) are the two key enzymes required for the production of lanosterol from an acyclic polyene precursor. Squalene epoxidase requires a supernatant protein factor (SPF) for activity. Classical purifications have been reported for rat liver SE and rat liver (SPF). Several OSC enzymes have been purified from plants, but the vertebrate enzymes have as yet eluded purification to homogeneity. Our goal is to purify, clone, and sequence these proteins from rat liver and then from human hepatoma cells. Furthermore, photoaffinity labels and irreversible enzyme-activated inhibitors recently discovered in our labs will enable us to selectively and covalently modify the active sites of both enzymes. First, we will synthesize selective inhibitors and radiolabeled substrates and photoaffinity labels for SPF, SE and OSC. Next, using these substrates, selective inhibitors and photoaffinity labels, we propose to purify these three proteins from rat liver. Rats will be induced to increased levels of cholesterol biosynthetic enzymes by ingestion of cholestyramine and an HMG-CoA reductase inhibitor or a SE epoxidase inhibitor. Enzymes will be purified by classical and affinity methods, using photoaffinity labeling and enzyme activity assays to monitor purification. Controlled trypsinization will be used to excise catalytically-active protein substructures from membrane proteins. Purified OSC and SE activity will be employed to obtain N-terminal and internal amino acid sequence data to design corresponding antisense oligonucleotide probes. PCR cloning will be employed to screen a rat liver cDNA library in lambda/gt11 to obtain full-length clones for these enzymes. cDNA probes from this system will then be used to identify SE and OSC cDNAs in a human hepatoma cell (Hep G2) cDNA library. A heterologous expression system will be developed to provide milligram quantities of the human proteins for studies of active site chemistry. Covalently-modified SE and OSC will be subjected to selected proteolysis to define the residues present in the active site.

角鲨烯环氧酶（SE）和氧化角鲨烯环化酶（OSC）是两个关键酶， 从无环化合物生产羊毛甾醇所需的酶 多烯前体。 角鲨烯环氧酶需要一个上清液蛋白 SPF（活动系数）。 已经报道了经典的纯化 大鼠肝脏SE和大鼠肝脏（SPF）。 几种OSC酶已经被 从植物中纯化出来，但脊椎动物的酶还没有消失。 纯化至均一。 我们的目标是提纯克隆测序 这些蛋白质来自大鼠肝脏，然后来自人类肝癌细胞。 此外，光亲和标记物和不可逆的酶激活的 我们实验室最近发现的抑制剂将使我们能够选择性地 并共价修饰两种酶的活性位点。 首先，我们将合成选择性抑制剂和放射性标记 用于SPF、SE和OSC的底物和光亲和标记物。 接下来，使用 这些底物、选择性抑制剂和光亲和标记物， 建议从大鼠肝脏中纯化这三种蛋白质。 大鼠将 诱导胆固醇生物合成酶水平升高， 摄入消胆胺和HMG-CoA还原酶抑制剂或SE 环氧酶抑制剂。 酶将通过经典和亲和纯化 方法，使用光亲和标记和酶活性测定， 监测净化。 将使用受控胰蛋白酶消化来切除 催化活性蛋白质的亚结构从膜蛋白。 将使用纯化的OSC和SE活性来获得N-末端和N-末端修饰。 内部氨基酸序列数据设计相应的反义 寡核苷酸探针。 将采用PCR克隆技术筛选大鼠 在λ/gt 11中的肝cDNA文库，以获得这些的全长克隆 内切酶 然后将来自该系统的cDNA探针用于鉴定SE 和人肝癌细胞（Hep G2）cDNA文库中的OSC cDNA。 一 将开发异源表达系统以提供毫克 用于活性部位化学研究的大量人类蛋白质。 共价修饰的SE和OSC将进行选定的蛋白水解 以确定活性位点中存在的残基。