TITLE	Characterization of 1-aminocyclopropane-1-carboxylate (ACC) oxidase in broccoli florets and from Escherichia coli cells transformed with cDNA of broccoli ACC oxidase
AUTHOR	Yuko Kasai Department of Biological Sciences, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan Hiroshi Hyodo Department of Biological Sciences, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan Yoshinori Ikoma Department of Citriculture, National Institute of Fruit Tree Science, Okitsu, Shimizu 424-0204, Japan Masamichi Yano Department of Citriculture, National Institute of Fruit Tree Science, Okitsu, Shimizu 424-0204, Japan
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ABSTRACT	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 CO2 in its activity besides ACC and O2 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.
KEYWORD	1-Aminocyclopropane-1-carboxylate (ACC) oxidase; Brassica oleracea; Broccoli florets; Escherichia coli; Senescence; Transformant;
ARTICLE INFO	Botanical Bulletin of Academia Sinica, Volume 39 Number 4 October 1998, page 225-230, 6 pages
PUBLISHER	Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China