Characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase in broccoli florets and from Escherichia coli cells transformed with cDNA of broccoli ACC oxidase

During senescence of broccoli (Brassica oleracea L. var. italica) florets (flower buds), the rate of ethylene production increased, almost paralleling the rapid increase in 1-aminocyclopropane-1-carboxylate (ACC) oxidase activity. The increase in ACC oxidase activity was strongly suppressed by the administration of cycloheximide, suggesting that it may have resulted from the de novo synthesis of enzyme protein. ACC oxidase extracted from broccoli florets exhibited the requirements for ferrous ion, ascorbate, and CO 2 in its activity besides ACC and O 2 as substrates. ACC oxidase was isolated and purified from Escherichia coli cells transformed with cDNA of broccoli ACC oxidase. The recombinant bacterial enzyme was very similar to that of native broccoli enzyme in its enzymatic properties. The molecular mass of the purified enzyme from the transformed cells was estimated to be 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The polyclonal antibody was raised in a rabbit against the purified enzyme isolated from the transformed bacteria, which was found to cross react with the enzyme of broccoli florets. Western blot analysis showed that ACC oxidase protein was detected at an almost similar level over the progressive stages of senescence.