The a-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-1 14 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/ celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the a-ketoglutarate transport protein catalyzed a phthalonate-sensitive a-ketoglutarate/a-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the a-ketoglutarate transport system as characterized in plant and animal mitochondria.The inner membrane of both mammalian and plant mitochondria contains specific carrier systems for the transport of different substrates (8,12,16). Among these systems, an aketoglutarate carrier has been characterized in mammals (18,21) and plants (9).Triton-extraction followed by chromatography on hydroxyapatite and celite has been used to purify several carriers from animal mitochondria to homogeneity (14). However, in plants, none of the mitochondrial metabolite carriers have been purified so far. Only the pyruvate carrier from castor bean mitochondria (4) and the dicarboxylate and glutamate/ aspartate carriers from pea leaf mitochondria (22, 23) have been partially purified. In this paper, we describe a partial purification of the a-ketoglutarate carrier from corn mitochondria using functional reconstitution as a monitor of the carrier activity during isolation. MATERIALS AND METHODS Plant Material and ChemicalsCorn seeds (Zea mays L., B 73) were surface-sterilized for 2 min in 1% (w/v) Isolation and Purification of Mitochondria Mitochondria were isolated from etiolated corn shoots and purified by rapid centrifugation through a 0.6 M mannitol "cushion" as described by Day and Hanson (5) with little modification. The grinding medium was 0.4 M mannitol, 10 mM Tris HCl, pH 8.0, 1 mM EDTA, 0.05% cysteine, and 0.1% BSA. In addition, a Braun mixer was used to disrupt the tissue rather than a mortar and pestle. The crude mitochondrial pellet was resuspended in a washing medium containing 0.3 M mannitol and 10 mm Tris HCl, pH 7.2. The supernatant, after centrifugation at 10OOg for 10 min, was gently underlaid with a cushion of 0.6 M mannitol and 10 mM Tris HCl, pH 7.2, and centrifuged at I0,000g for 20 min. Purification of the a-Ketoglutarate Transport ProteinCorn shoot mitochondria (15-18 mg protein/mL) were solubilized in 3% Triton X-114 (w/v), 20 mm Na2SO4, 10 mM Pipes, and 1 mM EDTA (pH 7.0). After 10 min at 0°C, the mixture was centrifuged at 140,000g for 20 min to obtain a clear supernatant referred to as the extract. Five hundred microliters of the extract (3.0-3.5 mg protein) supplemented with cardiolipin (6.0 mg/mL) were applied to cold hydroxyapatite columns ...
The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Cel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular m a s of 32 kD. When reconstituted i n liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, CDP, and CTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5'-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 pmol min-' mg-' protein at 25°C. The half-saturation constants and the corresponding inhibition constants were 17 PM for ATP, 26 p~ for ADP, 59 p~ for CTP, and 125 p~ for CDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between O and 15"C, and 22 kilojoule/mol between 15 and 35°C. Partia1 amino acid sequences showed that the purified protein was the product of the ANT-C1 gene sequenced previously (8. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).The inner membrane of plant mitochondria contains at least 10 specific carrier systems for the transport of metabolites (Day
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