Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria.
The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25 degrees C reached 2000 mumols/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).
Bisaccia
F
F
Department of Pharmaco-Biology, University of Bari, Italy.
De Palma
A
A
Prezioso
G
G
Palmieri
F
F
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002845:Chromatography; D002951:Citrates / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008293:Malates / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D014233:Tricarboxylic Acids / Q000378:metabolism*
D002352:Carrier Proteins; D002951:Citrates; D008081:Liposomes; D008293:Malates; D014233:Tricarboxylic Acids