McCormick J I, Johnstone R M
McGill University, Department of Biochemistry, Montreal, Quebec, Canada.
J Biol Chem. 1988 Jun 15;263(17):8111-9.
Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.
通过添加大豆卵磷脂囊泡、凝胶过滤和冻融循环，可以从溶解的艾氏腹水癌细胞质膜中重建钠依赖性氨基酸转运。用Ba2+沉淀去除磷脂酸（约占总脂质的10%）会使钠依赖性氨基酸转运的重建效率降低约73%，并使蛋白脂质体的囊泡内体积减少约43%。转运活性的丧失并非由于重建过程中特定蛋白质的排除。不含磷脂酸的脂质体通透性较低，达到溶质平衡分布所需的时间更长。在重建过程中添加卵磷脂酸，可以将转运活性和囊泡内体积恢复到经Ba2+沉淀的大豆卵磷脂蛋白脂质体。转运活性的恢复程度与囊泡内体积的变化成正比，并取决于磷脂酸的含量。用20%的磷脂酰丝氨酸或磷脂酰甘油替代磷脂酸会导致囊泡内体积增加，而氨基酸转运几乎没有增加或没有增加。用磷脂酶D处理经Ba2+沉淀的蛋白脂质体原位生成磷脂酸也能恢复转运。观察到的转运活性增加（9倍）伴随着囊泡内体积增加46%，这可能是由囊泡融合引起的。从纯磷脂酰胆碱：磷脂酰乙醇胺（1:1）混合物中成功重建钠依赖性氨基酸转运也需要磷脂酸，此时囊泡内体积仅有微小变化（约16%）。这些结果为磷脂酸对钠依赖性氨基酸转运重建的间接和直接作用提供了证据。间接作用是通过扩大囊泡内体积发生的，大囊泡显示出更高的转运速率。然而，也有证据表明磷脂酸对钠依赖性氨基酸转运体有特定作用，因为其他酸性脂质可能改变囊泡内体积而转运活性没有相应变化。
