The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies 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, characterized by a fast and independent binding of internal and external substrates.
Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria
DE PALMA, Annalisa;
2001-01-01
Abstract
The transport mechanism of the reconstituted ornithine/citrulline carrier purified from rat liver mitochondria was investigated kinetically. A complete set of half-saturation constants (K(m)) was established for ornithine, citrulline and H(+) on both the external and internal side of the liposomal membrane. The internal affinity for ornithine was much lower than that determined on the external surface. The exclusive presence of a single transport affinity for ornithine on each side of the membrane indicated a unidirectional insertion of the ornithine/citrulline carrier into liposomes, probably right-side-out with respect to mitochondria. Two-reactant initial velocity studies of the homologous (ornithine/ornithine) and heterologous (ornithine/citrulline) exchange reactions resulted in a kinetic pattern which is characteristic of a simultaneous antiport mechanism. This type of mechanism implies 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, characterized by a fast and independent binding of internal and external substrates.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
