Novel reconstitution and enzymatic assay of the mitochondrial tricarboxylate carrier.
The tricarboxylate carrier from beef liver mitochondria was reconstituted into liposomes using a protocol based on the absorption of Triton X-100 to hydrophobic Amberlite XAD-2 beads. The activity of the reconstituted carrier was determined spectroscopically by measuring the citrate/isocitrate exchange with an enzymatic assay. The Km for citrate obtained with this method was 35 microM and the Ki of 1,2,3-benzenetricarboxylate was 27 microM.
Lüthy
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Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Azzi
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D000818:Animals; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002951:Citrates / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D008081:Liposomes; D008930:Mitochondria, Liver / Q000378:metabolism*; D010713:Phosphatidylcholines; D010743:Phospholipids; D013367:Submitochondrial Particles / Q000378:metabolism*
D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D002951:Citrates; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; C020723:citrate-binding transport protein; C024990:asolectin