Purification and Properties of Amine Oxidase from Epicotyls of Pisum sativum

Abstract

A procedure has been developed for the purification of amine oxidase (E.C. 1.4.3.4) from etiolated pea epicotyls (Pisum sativum cv. Little Marvel). The enzyme is sensitive to copper chelating reagents and carbonyl reagents, but is not inhibited by sulfhydryl reagents. The purified enzyme has a molecular weight of 1.85 X 105, as determined by sedimentation equilibrium centrifugation, and has been shown to be specifically stimulated by phosphate.enzyme. In addition, the enzymes from bovine and porcine plasma have been found to contain pyridoxal phosphate (4, 23). The amine oxidase from porcine plasma has a molecular weight of 1.95 X 105 (5), and the amine oxidase from bovine plasma has a molecular weight of 1.7 x 10' (1). The enzyme from bovine plasma has a subunit molecular weight of 8.05 X 104(1).The study reported here describes a purification procedure for the amine oxidase from etiolated pea epicotyls, confirmation of copper and R-COR as functional groups in catalysis, the molecular weight as determined by ultracentrifugation, and the requirement of phosphate for maximal catalysis.Recent studies in this laboratory have indicated the possibility that IAA synthesis in Pisum could proceed through tryptamine and that the enzyme responsible for catalyzing this reaction could be purified (10, 14). As part of an investigation of IAA biosynthesis in Pisum, an enzyme which forms IAA from tryptamine was purified and found to be an amine oxidase. In view of the wide interest in amine oxidases from mammals and the one amine oxidase purified from a higher plant, it was decided to characterize this enzyme for a further understanding of the plant enzyme and for a comparison with the mammalian enzymes.Amine oxidase from plants was first purified and studied by Mann, who found that the enzyme was a pink, copper-containing protein which was sensitive to carbonyl reagents (12). The visible absorption spectrum of this enzyme had an absorbance maximum at 500 nm (13). This absorbance maximum could be shifted to lower wavelengths by incubation of the enzyme with substrate under anaerobic conditions or by incubation of the enzyme with copper-chelating reagents. These spectral shifts could be reversed by incubation under aerobic conditions in the first case and cupric ions in the second (9). The enzyme was found to be active in catalyzing the oxidative deamination of a wide variety of substrates (9). The purified enzyme had an s5,,0 of 7.7S, measured from sedimentation velocity centrifugation, and a molecular weight of 9.6 X 104, calculated from electron micrographs (9). Recently, Yamasaki et al. (24) have shown that the binding of oxygen to amine oxidase from peas is dependent upon amine concentration.Amine oxidase from bovine plasma has been studied extensively by Yasunobu and co-workers (21-23) who have found that the visible absorbance spectrum, copper content, and sensitivity to carbonyl reagents are similar to the plant

Multiple Amine Oxidases in Cucumber Seedlings

“…This would not be the case with AO-I, however. Finally, the substrate specificities of the two enzymes, although similar, are not exactly the same, and the Kmii values determined with several substrates are lower for AO-1 than those reported for the pea enzyme (13).…”

“…Michaelis constants and maximum velocities, relative to that obtained with TNH2, for several substrates are shown in Table III (13) suggested that the lower value reported by Hill and Mann (5) was the result of subunit dissociation during the procedures used for electron microscopy. This would not be the case with AO-I, however.…”

“…For kinetic studies involving different substrates, the peroxidase-coupled fluorometric assay of McGowan and Muir (13) was applied. The assay mixture contained 1.2 ml of buffer, 0.2 ml of substrate, 0.4 ml of enzyme, 0.1 ml of horseradish per- FIG.…”

Tryptamine in Zinc-Deficient Barley

“…Amineoxidase from the epicotyl of Pisum sativum was purified and studied by MCGOWAN and MUIR (14). The purified enzyme has a molecular weight of 1.85 x 10 5 • The Km for tryptamine was 1.6 mM.…”

Métabolisme et rôle des polyamines dans le développement de la plante

“…Les diamines oxydases sont particulierement, actives chez les legumineuses telles que Pisum sativum, Vicia faba, Lens esculatum et Phaseolus vulgaris (Me Gowan et Muir, 1971 ;Yanagisawa et al, 1981 ;Federico et al, 1985;Suzuki et al, 1990). Les DAO oxydent les fonctions amines primaires des diamines, Put et Cad, mais peuvent aussi oxyder les fonctions primaires des polyamines, Spd et Spm (Smith, 1985).…”