Cecchini G, Payne G S, Oxender D L
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Membr Biochem. 1978;2(1):149-58. doi: 10.3109/09687687809063863.
Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.
丙氨酸和亮氨酸的氨基酸转运系统已被重建到人工脂质小泡中。从艾氏腹水癌细胞中纯化的质膜小泡溶解于2%胆酸钠、1 mM二硫苏糖醇、0.5 mM乙二胺四乙酸中，该混合物可溶解约50%的膜蛋白。通过硫酸铵沉淀、凝胶过滤和DEAE-纤维素色谱相结合的方法对该溶解的蛋白质部分进行进一步纯化。在十二烷基硫酸钠凝胶上获得了一个含有约15条考马斯亮蓝染色带的组分。将该物质重建到脂质体中，初步结果表明丙氨酸和亮氨酸的转运依赖于钠梯度。此外，缬氨霉素诱导的钾扩散介导的生电梯度似乎进一步刺激了丙氨酸的摄取。
