Purification and Characterization of the Reconstitutively Active Citrate Carrier from Maize Mitochondria¹

Giuseppe Genchi, Anna Spagnoletta, Aurelio De Santis, Luisa Stefanizzi, and Ferdinando Palmieri^*

Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari and Consiglio Nazionale delle Ricerche Unit for the Study of Mitochondria and Bioenergetics, 70125 Bari, Italy (G.G., A.S., L.S., F.P.); Department of Pharmaco-Biology, University of Calabria, 87030 Cosenza, Italy (G.G.); and Department of Evolutionistic and Experimental Biology, University of Bologna, 40126 Bologna, Italy (A.D.S.)

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 min¹) or citrate/malate exchange (0.075 min¹). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, -ketoglutarate, oxaloacetate, and -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 min¹ mg¹ protein at 25°C.

Plant Physiol. (1999) 120: 841-848
Copyright Clearance Center:   0032-0889/99/120//08
© 1999 American Society of Plant Physiologists

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