Purification, properties and partial amino-acid sequence of 1-aminocyclopropane-1-carboxylic acid oxidase from apple fruits.

The enzyme which converts 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene, ACC oxidase, has been isolated from apple fruits (Malus x domestica Borkh. cv. Golden Delicious), and for the first time stabilized in vitro by 1,10-phenanthroline and purified 170-fold to homogeneity in a five-step procedure. The sodium dodecyl sulfate-denatured and native proteins have similar molecular weights (approx. 40 kDa) indicating that the enzyme is active in its monomeric form. Antibodies raised against a recombinant ACC oxidase over-produced in Escherichia coli from a tomato cDNA recognise the apple-fruit enzyme with high specificity in both crude extracts and purified form. Glycosylation appears to be absent because of (i) the lack of reactivity towards a mixture of seven different biotinylated lectins and (ii) the absence of N-linked substitution at a potential glycosylation site, in a sequenced peptide. Phenylhydrazine and 2-methyl-1-2-dipyridyl propane do not inhibit activity, indicating that ACC oxidase is not a prosthetic-heme iron protein. The partial amino-acid sequence of the native protein has strong homology to the predicted protein of a tomato fruit cDNA demonstrated to encode ACC oxidase.