The transport of amino acid across the membranes has great importance in cell metabolism. Specific experimental methodologies are required for measuring the vectorial reactions catalyzed by the membrane transporters. So far, the most widely used technique to study amino acid transport was the measure of amino acid flux in intact cell systems expressing a specific transporter. Some limitations in this procedure are caused by the presence of endogenous transporters and intracellular enzymes and by the inaccessibility of the intracellular compartment. Alternative experimental strategies which allow to reducing the interferences and improving the handling of the internal compartment would be useful to the amino acid transport knowledge.
An experimental protocol, which makes use of liposomes to study the transport of amino acid mediated by the glutamine/amino acid (ASCT2) transporter, solubilized from rat kidney brush borders, is described. The procedure is based on the reconstitution of the transporter in liposomes by removal of detergent from mixed micelles of detergent, solubilized protein, and phospholipid. The transport is assayed in the formed proteoliposomes measuring the Na+ dependent uptake of l-[3H]glutamine in antiport with internal l-glutamine. This method allows measuring the transport activity under well controlled experimental conditions and permits performing experiments which cannot be realized in intact cell systems.
Liposomes Reconstitution Transport Membrane Amino acids Glutamine
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This work was supported by the PRIN (Progetti di Ricerca Scientifica di Rilevante Interesse Nazionale) 2006 grant n. 2006054479 from MiUR (Ministero dell’Università e della Ricerca).
The author is indebted to Dr. Francesca Oppedisano, Dr. Lorena Pochini, and Dr. Michele Galluccio for help in preparing the manuscript.
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