Purification of the active mitochondrial tricarboxylate carrier by hydroxylapatite chromatography

Abstract

The mitochondrial tricarboxylate carrier has been extracted from rat liver mitochondria or SMP with Triton X-100, in the presence of 1,2,3-BTA and DPG, and partially purified by chromatography on HTP. The purified fraction, which also contains the ADP/ATP carrier and the phosphate carrier, after incorporation into liposomes catalyzes a 1,2,3-BTA-sensitive [14C]citrate/citrate exchange. The tissue and substrate specificity, the inhibitor sensitivity and the kinetic properties of citrate transport in liposomes are similar to those described for the citrate transport in mitochondria. The maximal rate of citrate exchange in the reconstituted system is 338 mumol X min-1 X g protein-1, at 30 degrees C and pH 7.0.