Purification and characterization of the reconstitutively active adenine nucleotide carrier from maize mitochondria.
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 Gel 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 mass of 32 kD. When reconstituted in 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, GDP, and GTP, 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 mumol min-1 mg-1 protein at 25 degrees C. The half-saturation constants and the corresponding inhibition constants were 17 microM for ATP, 26 microM for ADP, 59 microM for GTP, and 125 microM for GDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between 0 and 15 degrees C, and 22 kilojoule/mol between 15 and 35 degrees C. Partial amino acid sequences showed that the purified protein was the product of the ANT-G1 gene sequenced previously (B. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).
Genchi
G
G
Department of Pharmaco-Biology, University of Bari, Italy.
Ponzone
C
C
Bisaccia
F
F
De Santis
A
A
Stefanizzi
L
L
Palmieri
F
F
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D018520:Plant Shoots / Q000737:chemistry; D017421:Sequence Analysis; D003313:Zea mays / Q000737:chemistry*
D002352:Carrier Proteins; D006150:Guanine Nucleotides; D008081:Liposomes; D008565:Membrane Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases