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, alpha-ketoglutarate, oxaloacetate, and alpha-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 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C.
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
Genchi
A
A
Spagnoletta
A
A
De Santis
L
L
Stefanizzi
F
F
Palmieri
D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008081:Liposomes; D008928:Mitochondria / Q000737:chemistry*; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D008081:Liposomes; D010940:Plant Proteins; C020723:citrate-binding transport protein