PURIFICATION OF AMINOPHOSPHOLIPID FLIPPASE

磷脂在生物体内的分布是非随机的 膜。含有胆碱的磷脂被浓缩在 质膜外叶，而氨基磷脂 富含在细胞膜表面。这种不对称性是 部分由对钒和钙敏感的， 依赖于镁三磷酸腺苷的氨基磷脂转运体(一种“翻转酶”)。这个 我们工作的目标是了解 人红细胞膜中磷脂的不对称性 并确定不对称性的丧失在人类 心血管疾病。为此，我们正在进行三个项目： 负责氨基磷脂运输的蛋白质(S)正在被 从人红细胞膜中提纯。已有一个MgATPase 隔离的，具有许多与 氨基磷脂翻转酶。ATPase被钙抑制， 钒酸根试剂和巯基试剂。它被有选择地激活 磷脂酰丝氨酸(PS)；其他脂类几乎不支持或不支持ATPase 活动。PS具有高度的结构性和认可性 立体特异性。几乎所有对官能团的修饰 在PS上取消激活ATPase的能力。虽然L--和 D-丝氨酸类似物同样能激活ATPase活性，只有 天然产生的sn-1，2-甘油异构体支持ATPase活性。 最近，ATPase被重组为蛋白质脂质体，用于 脂类转运的研究。在未来的工作中，将提高抗体 针对这种蛋白质，它将被克隆，并进行生物物理研究 将执行翻转酶的结构和功能。众多合成材料 这项工作需要PS的磷脂和头基类似物。 用FAB-MS、MS/MS和Exact等方法对这些脂类进行了表征 质量测量。

Phospholipids are nonrandomly distributed across biological membranes. The choline-containing phospholipids are enriched on the plasma membrane outer leaflet, while the aminophospholipids are enriched on the cytofacial surface of the membrane. This asymmetry is maintained in part by a vanadate- and calcium-sensitive, MgATP-dependent, aminophospholipid transporter (a "flippase"). The goals of our work are to understand the mechanisms by which phospholipid asymmetry is maintained in human erythrocyte membranes and to determine the role that a loss of asymmetry plays in human cardiovascular disease. To this end we are pursuing three projects: The protein(s) responsible for aminophospholipid transport is being purified from human erythrocyte membranes. A MgATPase has been isolated that shares many characteristics in common with the aminophospholipid flippase. The ATPase is inhibited by calcium, vanadate and sulfhydryl reagents. It is selectively activated by phosphatidylserine (PS); other lipids support little or no ATPase activity. PS is recognized with high degree of structural and stereospecificity. Almost any modification to the functional groups on PS abolish the ability to activates the ATPase. Though L- and D-serine analogs activate ATPase activity equally well, only the naturally occuring sn-1,2-glycerol isomer supports ATPase activity. Recently, the ATPase has been reconstituted into proteoliposomes for studies of lipid transport. In future work, antibodies will be raised against the protein, it will be cloned, and biophysical studies of flippase structure and function will be performed. Numerous synthetic phospholipids and headgroup analogs of PS are required for this work. Each of these lipids has been characterized by FAB-MS, MS/MS and exact mass measurements.