Purification and Characterization of the Reconstitutively Active Citrate Carrier from Maize Mitochondria

The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5′-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min(−1)) or citrate/malate exchange (0.075 min(−1)). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, l-malate, succinate, malonate, glutarate, α-ketoglutarate, oxaloacetate, and α-ketoadipate and was inhibited by pyridoxal 5′-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2,3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10°C and 35°C; the half-saturation constant (K(m)) for citrate was 0.65 ± 0.05 mm and the maximal rate (V(max)) of the citrate/citrate exchange was 13.0 ± 1.0 μmol min(−1) mg(−1) protein at 25°C.