Detection of 1-aminocyclopropane-1-carboxylate oxidase activity in seeds of Stylosanthes humilis H.B.K

Abstract

The activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO) was characterized in seeds of the tropical legume Townsville stylo (Stylosanthes humilis) both in vitro (desalted extract of non-dormant seeds) and in vivo (entire dormant seeds). Optimum conditions for maximum in vitro ACO activity in a Trizma-HCl 100 mM buffered medium were: pH 7.0, temperature 32ºC and cofactors and co-substrate at the following concentrations: NaHCO 3 30 mM, sodium ascorbate 30 mM and FeSO 4 50 μM. Rates of in vitro reaction catalyzed by ACO were shown to be constant within the interval 15-150 minutes from the onset of the reaction. The apparent K m for in vitro ACO, as determined from the non-linear curve fitting to the Michaelis-Menten equation, was 156±8.3 μM ACC with a V max 5.4±0.08 mmol (ET) g -1 h -1 on a fresh matter (FM) basis. In vivo (control basal medium: HCl-KOH 10 mM pH 7.0, 30ºC, reaction time 15 hours) apparent K m was 230±27 μM ACC and V max 11.9±0.38 mmol (ET) g -1 .h -1 on a FM basis. These data suggest that the enzyme exhibits a relatively low affinity for the substrate. The well-known inhibitors of ACO activity, α-aminoisobutyric acid, salicylic and acetylsalicylic acids, n-propylgallate and cobaltous ions, were highly effective in inhibiting ACO activity of Townsville stylo seeds.