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 [H-3]ornithine uptake in proteoliposomes containing internal ornithine (ornithine/ornithine antiport mode) or as [H-3]ornithine efflux in the absence of external substrate (ornithine/H+ transport mode). 0.1 mM Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ strongly inhibited (more than 85%) the antiport; whereas Mn2+, Co2+ and Ni2+ inhibited less efficiently (25, 47 and 69%, respectively). The IC50 values of the transporter for the different metal ions ranged from 0.71 to 350 mu M. Co2+ and Ni2+ also inhibited the [H-3]ornithine efflux whereas Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ stimulated the [H-3]ornithine efflux. The stimulation of the [H-3]ornithine efflux by Cu2+ and Cd2+ (as well as by Pb2+, Hg2+ and Zn2+) 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 Cu2+, Pb2+, Hg2+, Cd2+ and Zn2+ with a population of SH groups, of the transporter, responsible for the inhibition of the physiological function; whereas the stimulation of [H-3]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 Ni2+, Co2+ or Mn2+ was caused by interaction with the substrate binding site, as indicated by the competitive or mixed inhibition.