Kinetic discrimination of two substrate binding sites of the reconstituted dicarboxylate carrier from rat liver mitochondria.
The kinetic interaction of various substrates and inhibitors with the dicarboxylate carrier from rat liver mitochondria was investigated using the isolated and reconstituted carrier protein. Due to their inhibitory interrelation the ligands could be divided into two classes: dicarboxylates, sulphate, sulphite and butylmalonate on the one hand and phosphate, thiosulphate and arsenate on the other. The mutual inhibition of substrates or inhibitors taken from one single class was found to be competitive, whereas the kinetic interaction of ligands when taken from the two different classes could be described as purely non-competitive. The half-saturation transport constants Km and the corresponding inhibition constants Ki of one single ligand, either used as substrate or as inhibitor, respectively, were found to be very similar. These kinetic data strongly support the presence of two different binding sites at the dicarboxylate carrier for the two different classes of substrates considering the external side of the reconstituted protein. When these two sites were saturated simultaneously with malate and phosphate, the turnover of the carrier was considerably reduced, hence indicating that a non-catalytic ternary complex is formed by the two substrates and the carrier molecule.
Indiveri
C
C
Department of Pharmaco-Biology, University of Bari, Italy.
Dierks
T
T
Krämer
R
R
Palmieri
F
F
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D013431:Sulfates / Q000378:metabolism / Q000494:pharmacology; D013447:Sulfites / Q000378:metabolism / Q000494:pharmacology; D013885:Thiosulfates / Q000378:metabolism / Q000494:pharmacology
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D008081:Liposomes; D008293:Malates; D010710:Phosphates; D013431:Sulfates; D013447:Sulfites; D013885:Thiosulfates; C030298:malic acid