Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit.
1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene. Following conventional column fractionation, the enzyme was purified 180-fold to near homogeneity with a specific activity of 20 nmol/(mg.min). This purified enzyme preparation migrated as a single protein band with an apparent molecular mass of 35 kDa on SDS/PAGE and 39 kDa on gel filtration. As in vivo, the purified enzyme required CO2 for activity. Removal of CO2 from the reaction mixture completely abolished the enzyme activity, while 0.5% atmospheric CO2 (0.15 mM in the medium) gave half-maximal activity. The purified enzyme displayed an absolute requirement for Fe2+ and ascorbate. The stoichiometry of the enzymatic reaction was determined: ACC + ascorbate + O2-->C2H4 + HCN + CO2 + dehydroascorbate + 2 H2O. A polyclonal antibody was raised against a synthetic tridecapeptide (PDLEEEYRKTMKE) whose sequence was deduced from the apple pAE12 cDNA [Dong, J. G., Olson, D., Silverstone, A. & Yang, S. F. (1992) Plant Physiol. 98, 1530-1531], which is homologous to tomato cDNAs encoding ACC oxidase. On a Western blot, this antibody specifically recognized the purified ACC oxidase protein. The amino acid composition of the purified enzyme agreed well with that deduced from the pAE12 sequence. When the protein was cleaved with CNBr and one of the peptide fragments was isolated and sequenced for 20 cycles, its sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115-134). When preclimacteric apple fruit was treated with ethylene, a parallel increase in in vivo and in vitro ACC oxidase activities was observed, and this increase was accompanied by a concomitant increase in the level of pAE12 transcript. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.
Dong
J G
JG
Mann Laboratory, Department of Vegetable Crops, University of California, Davis 95616.
Fernández-Maculet
J C
JC
Yang
S F
SF
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005030:Ethylenes / Q000378:metabolism; D005638:Fruit / Q000201:enzymology*; D008969:Molecular Sequence Data
D005030:Ethylenes; D002245:Carbon Dioxide; C036216:ethylene; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase