Kinetic mechanism of antiports catalyzed by reconstituted ornithine/citrulline carrier from rat liver mitochondria.
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.
Indiveri
C
C
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Italy.
Tonazzi
A
A
De Palma
A
A
Palmieri
F
F
D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D017920:Antiporters / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D002956:Citrulline / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism*; D009952:Ornithine / Q000737:chemistry*; D011522:Protons; D051381:Rats
D026923:Amino Acid Transport Systems, Basic; D017920:Antiporters; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D011522:Protons; C041356:ornithine transporter; D002956:Citrulline; D009952:Ornithine