A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Abstract

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K + exhibited high levels of Na +-dependent, α-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na +-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by d-glucose, d-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na +-dependent amino acid transport. The use of Na + in the reconstitution procedure led to formation of liposomes which displayed little Na +-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K + medium.