McCormick J I, Silvius J R, Johnstone R M
J Biol Chem. 1985 May 10;260(9):5706-14.
Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.
依赖钠离子的氨基酸转运可通过对与冻融循环偶联的解离质膜和大豆卵磷脂囊泡进行凝胶过滤来重建。所得的转运活性受到重建介质性质的显著影响。在钾离子存在下进行重建可形成活性脂质体，而在钠离子、锂离子或胆碱存在下进行重建则不能。钾离子脂质体的电子显微照片显示脂质体大小差异很大。对钾离子脂质体进行聚蔗糖密度梯度分级分离表明，最大的囊泡富含脂质，密度最低，且依赖钠离子的氨基酸转运水平最高。在钠离子存在下形成的脂质体捕获体积比钾离子脂质体小34%，且缺乏大囊泡群体。在钾离子存在下进行第二次冻融可恢复钠离子脂质体的活性，此时钠离子脂质体含有大的低密度活性囊泡。对冻融诱导的囊泡脂质混合进行荧光测量表明，钠离子脂质体中缺乏大囊泡是由于钠离子在冻融过程中抑制了脂质囊泡融合事件。大囊泡部分富含一种125 kDa的肽。该肽是否是中性氨基酸转运系统的一部分尚未确定。
