Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria.
The 2-oxoglutarate carrier from the inner membrane of bovine heart mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted with egg yolk phospholipid vesicles by the freeze-thaw-sonication technique. In the reconstituted system the incorporated 2-oxoglutarate carrier catalyzed a first-order reaction of 2-oxoglutarate/2-oxoglutarate exchange. The substrate affinity for 2-oxoglutarate was determined to be 65 +/- 18 microM (15 determinations) and the maximum exchange rate at 25 degrees C reaches 4000-22,000 mumol/min per g protein, in dependence of the particular reconstitution conditions. The activation energy of the exchange reaction is 54.3 kJ/mol. The transport is independent of pH in the range between 6 and 8. When the first fraction of the hydroxyapatite/celite column eluate was used for reconstitution, besides the 2-oxoglutarate/2-oxoglutarate exchange, a significant activity of unidirectional uptake was observed. This activity may be due to a population of the carrier protein which is in a different state.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008081:Liposomes; D026901:Membrane Transport Proteins; D011510:Proteolipids; C021723:oxoglutarate translocator; C020485:proteoliposomes