Purification and Characterization of the Reconstitutively Active Citrate Carrier from Maize Mitochondria1
| Autor: | Aurelio De Santis, Giuseppe Genchi, Ferdinando Palmieri, Anna Spagnoletta, Luisa Stefanizzi |
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| Rok vydání: | 1999 |
| Předmět: |
Chromatography
Molecular mass biology Physiology Biological Transport Plant Science Zea mays Mersalyl Mitochondria chemistry.chemical_compound Malonate chemistry Liposomes Genetics Cardiolipin biology.protein Citrate synthase Carrier Proteins Citric acid Pyridoxal Citrate test Research Article Plant Proteins |
| Zdroj: | Plant Physiology. 120:841-848 |
| ISSN: | 1532-2548 0032-0889 |
| Popis: | 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, andp-hydroxymercuribenzoate (but notN-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 (Km) for citrate was 0.65 ± 0.05 mm and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 ± 1.0 μmol min−1 mg−1 protein at 25°C. |
| Databáze: | OpenAIRE |
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