Cecchini G, Payne G S, Oxender D L
J Supramol Struct. 1977;7(3-4):481-7. doi: 10.1002/jss.400070317.
Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.
已通过用0.5% Triton X-100提取或用2%胆酸盐提取，从艾氏腹水癌细胞的质膜中获得了可溶蛋白组分。通过硫酸铵沉淀和柱色谱相结合的方法对可溶蛋白组分进行了部分纯化。在Triton X-100溶解的膜组分中检测到了亮氨酸结合活性。亮氨酸结合活性通过平衡透析法测定，高水平的亮氨酸或苯丙氨酸可使其饱和，且不受丙氨酸的强烈影响。这些特性与先前鉴定为L系统的特性相似。此外，对胆酸盐提取的蛋白组分进行了部分纯化，并将其重构到脂质体中。在重构的囊泡中可以证明丙氨酸和亮氨酸的钠依赖性摄取。浓缩摄取依赖于钠梯度。在存在钠的情况下，缬氨霉素介导的钾扩散产生的膜电位也刺激了重构脂质体中的氨基酸转运。
