Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria

The transport mechanism of the reconstituted ornithine/citrulline carrier p urified from rat liver mitochondria was investigated kinetically. A complet e set of half-saturation constants (K-m) was established for ornithine, cit rulline and Hf on both the external and internal side of the liposomal memb rane. The internal affinity for ornithine was much lower than that determin ed on the external surface. The exclusive presence of a single transport af finity for ornithine on each side of the membrane indicated a unidirectiona l insertion of the ornithine/citrulline carrier into liposomes, probably ri ght-side-out with respect to mitochondria, Two-reactant initial velocity st udies of the homologous (ornithine/ornithine) and heterologous (ornithine/c itrulline) exchange reactions resulted in a kinetic pattern which is charac teristic of a simultaneous antiport mechanism. This type of mechanism impli es that the carrier forms a ternary complex with the substrates before the transport reaction occurs. A quantitative analysis of substrate interaction revealed that rapid-equilibrium random conditions were fulfilled, characte rized by a fast and independent binding of internal and external substrates . (C) 2001 Elsevier Science B.V. All rights reserved.