Chemical modification of the mitochondrial ornithine/citrulline carrier by SH reagents: effects on the transport activity and transition from carrier to pore-like function

Abstract

The transport activity of the purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria was correlated to modification of its sulfhydryl groups by various reagents. Both the ornithine/ornithine (antiport) and the ornithine/H + (unidirectional) transport modes catalysed by the ornithine/citrulline carrier were inhibited by methanethiosulfonates, mercurial reagents, N-ethylmaleimide (NEM) and 5,5′-dithiobis(2-nitrobenzoate) (DTNB). The treatment of the ornithine/citrulline carrier with mercurial reagents, at concentrations above 5 μM, caused the induction of an additional (pore-like) transport mode, characterized by loss of substrate specificity and a transport activity higher than that of the unmodified carrier. The S–S forming reagent Cu 2+-phenanthroline inhibited the transport catalysed by the carrier, indicating the presence of close sulfhydryl groups. The effect of consecutive addition of the various reagents revealed a peculiar aspect of the ornithine/citrulline carrier, i.e. the presence of three distinct populations of sulfhydryl groups. The first was responsible for the inhibition of the physiological transport modes by methanethiosulfonates, NEM and DTNB and low concentrations (<5 μM) of mercurials; the second population was responsible for the transition to the pore-like activity induced by higher concentrations (>5 μM) of mercurials; the third population was involved in S–S bridge formation.