Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers.
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.
Spagnoletta
Anna
A
Department of Pharmaco-Biology, University of Calabria, Cosenza, Italy.
De Santis
Aurelio
A
Palmieri
Ferdinando
F
Genchi
Giuseppe
G
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
D008081:Liposomes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases