REGULATION OF PHOSPHOLIPID TRANSFER BETWEEN MEMBRANES

磷脂分子在膜之间的自发转移发生 通过可溶性单体对流通过水相。 平衡 磷脂单体在水相中和 膜相与磷脂转移速率相关。 为 因此，人们认为磷脂的水溶性低， 以测定磷脂在膜之间的转移速率。 在 这一建议的证据是，提出了自发的膜间 磷脂转移受到大的活化自由能的限制， 单体膜的解离和缔合与，但 而不是由分配平衡决定的。 这个项目的目标是 为了确定不同磷脂中该活化能的值， 单体-膜系统及其在调节磷脂转移中的作用 在膜之间。 基于共振能量转移（RET）的技术 将用于监测荧光标记的 磷脂囊泡之间的磷脂。 测量 的解离速率常数的温度依赖性 荧光磷脂的RET技术和测量 将通过离心技术进行单体-囊泡分配平衡， 组合以构建囊泡中单体的相对能量图， 水和过渡态，包括相对贡献 焓和熵 的转移速率常数的依赖性 将测量压力和囊泡大小并用于配制 解离-缔合障碍的机械描述。 如果 是磷脂单体-膜大自由能垒 解离和缔合，然后磷脂转移蛋白可以起作用， 为了通过降低该高度来促进单体对流的速率， 屏障 因此，这一机制的相对重要性， 与航天飞机载体机制相比， 两种牛肝脂质转移蛋白（磷脂酰胆碱特异性 转移蛋白和非特异性磷脂转移蛋白）。 测量 蛋白质依赖性跨透析膜转移速率 因为动力学建模将用于此目的。

The spontaneous transfer of phospholipid molecules between membranes occurs by soluble monomer convection through the aqueous phase. The equilibrium partition between phospholipid monomers in the aqueous phase and in the membrane phase is correlated with the rate of phospholipid transfer. For this reason, the low aqueous solubility of phospholipids has been assumed to determine the rate of transfer of phospholipids between membranes. In this proposal evidence is presented that spontaneous intermembrane phospholipid transfer is limited by a large free energy of activation for monomer-membrane dissociation and association that is correlated with, but not determined by, the partition equilibrium. The goal of this project is to determine the value of this activation energy in different phospholipid monomer-membrane systems and its role in regulating phospholipid transfer between membranes. A technique based on resonance energy transfer (RET) will be used to monitor the rate of transfer of fluorescently labeled phospholipids between phospholipid vesicles. Measurements of the temperature dependence of the dissociation rate constant for the fluorescent phospholipids by the RET technique and measurements of the monomer-vesicle partition equilibrium by a centrifugation technique will be combined to construct a relative energy diagram for a monomer in vesicles, water, and transition states, that includes the relative contributions of enthalpy and entropy. The dependence of the transfer rate constants on pressure and vesicle size will be measured and used to formulate a mechanistic description of the dissociation-association barrier. If there is a large free energy barrier for phospholipid monomer-membrane dissociation and association, then phospholipid transfer proteins may act to facilitate the rate of monomer convection by reducing the height of this barrier. Consequently, the relative importance of this mechanism as compared to a shuttle-carrier mechanism will be determined for the activity of two bovine liver lipid transfer proteins (phosphatidylcholine-specific transfer protein and nonspecific phospholipid transfer protein). Measuring the rate of protein-dependent transfer across a dialysis membrane as well as kinetic modeling will be used for this purpose.