CHARACTERIZATION OF AN ENDOGENOUS INHIBITOR OF THE ENZYMATIC CONVERSION OF THE ENZYMATIC CONVERSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID TO ETHYLENE IN CARNATION PETALS

Abstract

The membrane-bound enzymatic conversion of 1 -aminocyclopropane-1 -carboxylic acid (ACC) to ethylene was demonstrated in carnation petals by Mayak et al. (1981), who also reported the presence of an endogenous, soluble inhibitor of this reaction. The aim of the present work was to characterize this inhibitor. In a standard reaction system using ACC as a substrate and intact or detergent-solubilized microsomal membranes from carnation petals as a source of enzymatic activity, ethylene production could be inhibited by the addition of various amounts of petal extract. Similar effects on ethylene formation were found when petals were treated with extract solution before being fed with ACC. The inhibition was reversible and competitive, with Ki equivalent to 0.8 mg of the original fresh weight. The presence of the inhibitor did not change the optimal pH of the reaction, but the extent of inhibition was highly dependent on pH, maximum inhibition occurring at pH 8.0. No binding to a cation exchanger was detected. The inhibitor was, however, adsorbed in a pH-dependent manner onto an anion exchanger, DEAE-Sephadex, with no further changes in charge below pH 3.2. The inhibitor was dialyzable, and gel filtration indicated that it had a low molecular weight. In addition, the inhibitor was stable when exposed to pH extremes and heat, it was not precipitated by trichloroacetic acid (TCA), and could be completely separated from proteins by gel chromatography; these data indicate its nonproteinaceous nature. The results of ultraviolet, infrared, and fluorescence spectroscopy of the semipurified material, together with the fact that it binds to polyvinylpolypynolidone, support our assumption that the inhibitory activity of carnation petal extract may be associated with phenolic compounds.