title: Effects of heavy metal cations on the mitochondrial ornithine/citrulline transporter reconstituted in liposomes.
abstract: The effect of heavy metal cations on the mitochondrial ornithine/citrulline transporter was tested in proteoliposomes reconstituted with the protein purified from rat liver. The transport activity was measured as [(3)H]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [(3)H]ornithine efflux in the absence of external substrate (ornithine/H(+) transport mode). 0.1 mM Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) strongly inhibited (more than 85%) the antiport; whereas Mn(2+), Co(2+) and Ni(2+) inhibited less efficiently (25, 47 and 69%, respectively). The IC(50) values of the transporter for the different metal ions ranged from 0.71 to 350 μM. Co(2+) and Ni(2+) also inhibited the [(3)H]ornithine efflux whereas Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) stimulated the [(3)H]ornithine efflux. The stimulation of the [(3)H]ornithine efflux by Cu(2+) and Cd(2+) (as well as by Pb(2+), Hg(2+) and Zn(2+)) was not prevented by NEM and was reversed by DTE. These features indicated that the inhibition of the antiport was due to the interaction of the Cu(2+), Pb(2+), Hg(2+), Cd(2+) and Zn(2+) with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [(3)H]ornithine efflux was due to the induction of a pore-like function of the transporter caused by interaction of cations with a different population of SH groups. Differently, the inhibition of the ornithine transporter by Ni(2+), Co(2+) or Mn(2+) was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.
authors.affiliation: CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, Bari, Italy. a.tonazzi@ibbe.cnr.it
authors.forename: Annamaria
authors.initials: A
authors.lastname: Tonazzi
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002412:Cations / Q000494:pharmacology*; D002956:Citrulline / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008081:Liposomes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D019216:Metals, Heavy / Q000494:pharmacology*; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D008958:Models, Molecular; D009952:Ornithine / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats
chemical_list: D026923:Amino Acid Transport Systems, Basic; D002412:Cations; D008081:Liposomes; D019216:Metals, Heavy; C041356:ornithine transporter; D002956:Citrulline; D009952:Ornithine