Effects of heavy metal cations on the mitochondrial ornithine/citrulline transporter reconstituted in liposomes

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 mu M. 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.