PURIFICATION OF THE AMINOPHOSPHOLIPID FLIPPASE

氨基磷脂翻转酶的纯化

• 批准号: 2184620
• 负责人: David L Daleke
• 金额: $ 10.27万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184620
• 关键词: adenosinetriphosphatase; affinity chromatography; binding proteins; chemical synthesis; erythrocyte membrane; erythrocytes; gel filtration chromatography; human tissue; laboratory rat; lipid transport; liposomes; membrane lipids; membrane reconstitution /synthesis; membrane transport proteins; phosphatidylcholines; phosphatidylserines; phospholipids; protein purification

Phospholipids in biological membranes are asymmetrically oriented across the bilayer, the choline containing phospholipids are localized primarily in the membrane outer monolayer and the amine-containing phospholipids, phosphatidylethanolamine and phosphatidylserine (PS), are sequestered in the membrane inner monolayer. Loss of PS asymmetry is associated with cell senescence and contributes to thromboses in heart disease, stroke, and diabetes. Phospholipid asymmetry in maintained in part by ATP-dependent transport of aminophospholipids from the membrane outer to inner surface. Aminophospholipid transport, or "flippase", activity has been well characterized in the plasma membrane and internal organelles of a variety of cells. Transport requires Mg2+-ATP and is inhibited by vanadate, sulfhydryl or arginine modification, and calcium. Recently, two proteins have been proposed as candidate transporters: a vanadate-sensitive PS-dependent ATPase (about 120 Kda) and a protein that reacts with sulfhydryl and PS affinity probes (32 Kda). A preliminary purification of the vanadate-sensitive, PS-dependent ATPase suggests that the flippase may be a complex of several proteins. The goal of the proposed work is to identify, purify and reconstitute the aminophospholipid flippase. Three specific aims will be addressed: 1) Improved sulfhydryl and photoaffinity (carbene and radical) PS analogs will be synthesized. These affinity lipids will be used to identify PS-binding and sulfhydryl-containing proteins in human erythrocytes. 2) Candidate aminophospholipid transporters will be purified from erythrocytes and rat brain using standard chromatographic and novel affinity methods. A unique PS affinity matrix will be constructed to selectively purify PS-binding proteins. The partially purified ATPase will be fractionated by gel-filtration and additional affinity purification methods. Distinguishing characteristics of PS transport, including PS and ATP binding, will be determined. Antibodies will be raised against purified proteins and will be used as probes of the structure, function, identification, and eventual molecular cloning of the flippase. 3) Purified candidate transporters will be reconstituted into liposomes and ATP-dependent transport activity will be measured. Flippase activity will be determined by measuring changes in PS transmembrane distribution with an enzymatic assay based on the PS-dependent activation of protein kinase C and by fluorescence resonance energy transfer between labeled phospholipids. The proposed studies may result in the identification and functional reconstitution of the aminophospholipid flippase. Future molecular biological and biophysical studies of the structure and function of the flippase will be made possible. New materials and methods developed in the course of this work will also find use in other membrane studies. This work will provide an understanding of the molecular mechanisms controlling membrane asymmetry that may lead to new strategies for the prevention of vascular disease.

生物膜中的磷脂是不对称取向的， 双层，含胆碱的磷脂 主要在膜外单层和含胺的 磷脂、磷脂酰乙醇胺和磷脂酰丝氨酸（PS）， 隔离在膜内单层中。PS不对称性的丧失是 与细胞衰老有关，并导致心脏血栓形成 中风和糖尿病。磷脂不对称性维持在 部分通过ATP依赖性的氨基磷脂从膜上转运 从外表面到内表面。氨基磷脂转运，或“翻转酶”， 活性已在质膜和内部被很好地表征 各种细胞的细胞器。运输需要Mg 2 +-ATP， 被钒酸盐、巯基或精氨酸修饰和钙抑制。 最近，已经提出两种蛋白质作为候选转运蛋白： 钒酸盐敏感的PS依赖性ATP酶（约120 Kda）和一种蛋白质 与巯基和PS亲和探针反应（32 Kda）。一 钒酸盐敏感的PS依赖性ATP酶的初步纯化 这表明翻转酶可能是几种蛋白质的复合物。的 拟议工作的目标是识别、纯化和重组 氨基磷脂翻转酶将处理三个具体目标： 1)改进的巯基和光亲和性（卡宾和自由基）PS 将合成类似物。这些亲和脂质将用于 鉴定人PS结合和含巯基蛋白质 红细胞 2)候选氨基磷脂转运蛋白将从 红细胞和大鼠脑，使用标准色谱和新的 亲和方法将构建独特的PS亲和矩阵， 选择性纯化PS结合蛋白。部分纯化的ATP酶 将通过凝胶过滤和额外的亲和性进行分级 纯化方法PS运输的显著特征， 包括PS和ATP结合。抗体将 针对纯化的蛋白质提出，并将用作探针的 结构、功能、鉴定和最终的分子克隆 翻转酶 3)将纯化的候选转运蛋白复溶至脂质体中 并测量ATP依赖性转运活性。翻转酶活性 将通过测量PS跨膜分布的变化来确定 用基于PS依赖性蛋白质活化的酶测定法 激酶C与通过荧光共振能量转移标记的 磷脂 拟议的研究可能会导致识别和功能 氨基磷脂翻转酶的重建。未来分子 生物学和生物物理学研究的结构和功能， 翻转酶将成为可能。开发的新材料和方法 这项工作的过程也将在其他膜研究中得到应用。 这项工作将提供一个分子机制的理解 控制膜的不对称性，这可能会导致新的战略， 预防血管疾病。