title: Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers.
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. Vmax of the reconstituted ATP/ATP exchange was determined to be 0.53 micromol/min per mg protein at 25 degrees C. The half-saturation constant Km and the corresponding inhibition constant Ki were 20.4 microM for ATP and 45 microM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30 degrees 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.
authors.affiliation: Department of Pharmaco-Biology, University of Calabria, Cosenza, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Spagnoletta
authors.forename: Aurelio
authors.initials: A
authors.lastname: De Santis
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Genchi
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D006368:Helianthus / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D008081:Liposomes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases