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Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs. Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery.

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The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-1 00 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAESephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.In liver, two enzymes of the urea cycle, ornithine transcarbamylase and carbamyl phosphate synthetase I, are located in the mitochondrial matrix, while the other enzymes are cytoplasmic. Thus, for the operation of the urea cycle, ornithine must pass from the cytosol to the mitochondrial matrix where it is carbamylated to citrulline, and citrulline must leave the mitochondria in order to regenerate ornithine.The presence of an ornithine/citrulline carrier in the inner mitochondrial membrane has already been predicted in 1972 from theoretical and metabolic considerations [l]. However, the experimental evidence supporting the existence of this carrier in intact mitochondria is only circumstantial and not as definitive as for other mitochondrial metabolite carriers (for a review see [2 and 31). Based on the osmotic swelling of rat liver mitochondria, Gamble and Lehninger [4] [7], using rat liver mitochondria loaded with citrulline, presented evidence for the existence of an ornithine/citrulline-exchange system. These authors observed the uptake of ornithine in citrulline-loaded mitochondria and citrulline efflux from these mitochondria upon addition of ornithine or lysine. In a subsequent study 181, no evidence could be obtained for an ornithine/citrulline or lysine/citrulline exchange, whereas it was concluded that ornithine and lysine are taken up by rat liver mitochondria in exchange for H'. Recently, in rat kidney mitochondria, an The final proof for the existence of a particular carrier protein is its isolation. So far, an ornithine-transport protein has only been reconstituted into liposomes from a Lubrol WX extract of rat liver mitochondria [lo]. In this paper, we describe the isolation and purification of the ornithine/citrulline carrier from rat liver mitochondria using functional reconstitution as a monitor of the carrier activity during the isolation procedure. MATERIALS AND METHODS MaterialsHydroxyapatite (Bio-Gel HTP) was purchased from BioRad, Celite 535 from Roth, Amberlite XAD-2 from Fluka, DEAE-Sephacel, Sephadex PD-10, Sephadex G-50 and Sephadex G-75 from Pharmacia, L-[ ...