Abstract
The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5′-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5′-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V max of the reconstituted ATP/ATP exchange was determined to be 0.53 μmol/min per mg protein at 25°C. The half-saturation constant K m and the corresponding inhibition constant K i were 20.4 μM for ATP and 45 μM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.
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Spagnoletta, A., De Santis, A., Palmieri, F. et al. Purification and Characterization of the Reconstitutively Active Adenine Nucleotide Carrier from Mitochondria of Jerusalem Artichoke (Helianthus Tuberosus L.) Tubers. J Bioenerg Biomembr 34, 465–472 (2002). https://doi.org/10.1023/A:1022570226209
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DOI: https://doi.org/10.1023/A:1022570226209