PURIFICATION OF THE AMINOPHOSPHOLIPID FLIPPASE

生物膜中的磷脂是不对称取向的。 双层，含胆碱的磷脂被定位 主要是在膜的外单分子层和含胺的 磷脂、磷脂酰乙醇胺和磷脂酰丝氨酸(PS)是 被隔离在膜内单层中。PS不对称性的损失是 与细胞衰老有关，并导致心脏血栓形成 疾病、中风和糖尿病。磷脂不对称性在体内的维持 部分通过依赖于ATP的氨基磷脂从膜上转运 从外到内的表面。氨基磷脂运输，或“翻转酶”， 在质膜和内部都有很好的活性特征 各种细胞的细胞器。运输需要Mg2+-ATP和IS 被钒酸盐、巯基或精氨酸修饰和钙抑制。 最近，有两种蛋白质被认为是候选的转运蛋白：a 钒酸盐敏感的PS依赖的ATPase(约120kDa)和一种蛋白质 它与巯基和PS亲和探针(32kda)反应。一个 钒酸盐敏感型PS依赖型ATPase的初步纯化 这表明该翻转酶可能是由几种蛋白质组成的复合体。这个 拟议工作的目标是识别、提纯和重组 氨基磷脂翻转酶。将解决三个具体目标： 1)改进了巯基和光亲和(卡宾和自由基)PS 类比将被合成。这些亲和力脂质将被用于 鉴定人PS结合蛋白和含硫蛋白 红血球。 2)候选氨基磷脂转运体将从 标准层析和新型层析技术在红细胞和大鼠脑中的应用 亲和力方法。将构建唯一的PS亲和力矩阵以 选择性纯化PS结合蛋白。部分纯化的ATPase 将通过凝胶过滤和额外的亲和力进行分级 纯化方法。PS传输的区别特征， 包括PS和ATP的结合，将被确定。抗体将会是 针对纯化的蛋白质而提纯，将用作 的结构、功能、鉴定和最终的分子克隆 这是一种翻转。 3)纯化的候选转运蛋白将被重组为脂质体 并将测量依赖于ATP的运输活动。翻转酶活性 将通过测量PS跨膜分布的变化来确定 以PS依赖的蛋白质激活为基础的酶分析 并通过荧光共振能量转移在标记的 磷脂。 拟议的研究可能导致识别和功能 氨基磷脂翻转酶的重组。未来分子 对细胞的结构和功能的生物学和生物物理研究 翻转酶将成为可能。开发的新材料和新方法 这项工作的过程也将在其他膜研究中使用。 这项工作将提供对分子机制的理解。 控制膜的不对称性，可能导致新的策略 预防血管疾病。

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.