Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick-pea (Cicer arietinum) seeds, which is dependent upon ethylene synthesis, a crude extract containing authentic ACC oxidase (ACCO) activity was isolated in soluble form from the embryonic axes of seeds germinated for 24 h. Under our optimal assay conditions (200 mM HEPES at pH 7.0, 4 μM FeSO 4 , 6 mM Na-ascorbate, 1 mM ACC, 20% O 2 , 3% CO 2 , and 10% glycerol) this enzyme was 5-fold more active than under the conditions we used initially in the present work. The enzyme has the following K m : 28 μM for ACC (approximately 4-fold less than in vivo), 1.2% for O 2 (in the presence of an optimal CO 2 concentration of 3%), and 1% for CO 2 in the presence of O 2 (20%). The enzyme is inhibited by phenanthroline (PNT) (specific chelating agent of ferrous ion), and competitively inhibited (K i = 0.5 mM) by 2-aminoisobutyric acid (AIB), and the enzymatic activity was not detectable in the absence of CO 2 . Under optimal assay conditions, the enzyme has two optimum temperatures (28 °C and 35°C) and is inhibited by divalent metal cations (Zn 2+ ≥ Co 2+ > Ni 2+ > Cu 2+ > Mn 2+ Mg 2+ ) and by sali cylic acid, propylgallate, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dinitrophenol (DNP), and Na-benzoate. The in vitro ACCO activity which we recovered in soluble form is equivalent to approximately 80-85% of the apparent activity evaluated in vivo