The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate-citrate exchange, while HgCl2 and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH2 residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.
Citrate uniport by mitochondrial tricarboxylate carrier: a basis to a new hipothesys for the transport mechanism
DE PALMA, Annalisa;SCALERA, Vito Dom. E.;
2003-01-01
Abstract
The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate-citrate exchange, while HgCl2 and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH2 residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
