REGULATION OF PHOSPHOLIPID TRANSFER BETWEEN MEMBRANES

本提案中提出的实验针对的是两个 主要目标。第一是继续研究脂质转移是如何 蛋白质促进膜之间的脂质运动，第二 是为了研究磷脂表面的程度和机制 高密度脂蛋白与成纤维细胞之间的转移--即磷脂 除了完整的高密度脂蛋白的转移外，还会发生的转移 粒子。这些研究主要集中在非特异性脂质转移上。 蛋白质(NsLTP)，因为它已经被广泛研究并被很好地 特点，但其作用机制和生理 功能是未知的。虽然它是从肝脏胞浆中分离出来的 并具有独特的刺激膜间转移的能力 在广泛的脂质分子中，它不是正常所必需的 细胞内的脂质代谢，因此其功能可能是 来转移血浆中的脂类。 我们实验室开发了一种使用荧光的新技术- 标记的磷脂首次证明 磷脂与nsLTP结合。结合动力学的研究 提出了一种结合-释放的载体模型 NsLTP刺激的膜间转移，其中nsLTP发生碰撞 与供体膜表面结合磷脂分子，以及 将其从膜上移除。结合磷脂迅速 与水相平衡，游离脂或 蛋白质结合的脂类与受体膜重新结合。这 假说将得到检验，膜和脂质的性质 将对决定nsLTP有效性的因素进行表征。 这项建议的第二个目标是研究地表迁移。 高密度脂蛋白颗粒和人成纤维细胞之间的磷脂。 尽管高密度脂蛋白和细胞之间的胆固醇运动 经过广泛研究，人们对磷脂转移知之甚少。 然而，磷脂酰胆碱向高密度脂蛋白的转移是 从外周细胞中去除胆固醇，因为大多数 胆固醇由高密度脂蛋白以胆固醇酯的形式携带。 这需要磷脂酰胆碱酰基的酶转移 与胆固醇相关的链条。这种高密度脂蛋白磷脂的来源不是 为人所知。在这一点上，转移的速度和机制 成纤维细胞与高密度脂蛋白之间的荧光标记磷脂 被调查以确定是自发的还是nsLTP- 刺激的磷脂转移可能在生理上提供 高密度脂蛋白磷脂的重要来源。

The experiments presented in this proposal are directed towards two major goals. The first is to continue to study how lipid transfer proteins enhance lipid movement between membranes, and the second is to study the extent of and mechanism for phospholipid surface transfer between HDL and fibroblasts--that is, phospholipid transfer that occurs in addition to transfer of intact, HDL particles. These studies focus on the nonspecific lipid transfer protein (nsLTP) because it has been studied extensively and is well characterized, yet its mechanism of action and physiological function are not known. Although it is isolated from liver cytosol and has the unique ability to stimulate the intermembrane transfer of a wide range of lipid molecules, it is not essential to normal intracellular lipid metabolism, and therefore its function may be to transfer lipids in the plasma. A new technique developed in our laboratory using fluorescent- labeled phospholipids has demonstrated for the first time phospholipid binding to nsLTP. Studies of the binding kinetics suggest a bind-and-release carrier model for the mechanism of nsLTP-stimulated intermembrane transfer, in which nsLTP collides with the donor membrane surface, binds a phospholipid molecule, and removes it from the membrane. The bound phospholipid rapidly equilibrates with the water phase and either the free lipid or protein-bound lipid reassociates with the acceptor membrane. This hypothesis will be tested and the membrane and lipid properties that determine nsLTP effectiveness will be characterized. The second goal of this proposal is to study the surface transfer of phospholipids between HDL particles and human fibroblasts. Although cholesterol movement between HDL and cells has been studied extensively, little is known about phospholipid transfer. Yet phosphatidylcholine transfer to HDL is a requirement for cholesterol removal from peripheral cells since the majority of cholesterol is carried by HDL in the form of cholesterol esters which requires the enzymatic transfer of a phosphatidylcholine acyl chain to cholesterol. The source of this HDL phospholipid is not known. In this light, the rate and mechanism of transfer of fluorescent-labeled phospholipids between fibroblasts and HDL will be investigated to determine whether spontaneous or nsLTP- stimulated phospholipid transfer may provide a physiologically important source of HDL phospholipid.