Further properties of the diamine oxidase of pea seedlings

Abstract

Purified preparations of pea-seedling diamine oxidase catalyzed the oxidation not only of aliphatic diamines but also that of aliphatic monoamines, phenylalkylamines, histamine, spermidine, agmatine, and the amino acids lysine and ornithine. The diamine-oxidase prep-arations were pink solutions with maximum absorption, in the visible region, at about 500 m[mu]. When substrate was added under anaerobic conditions the color changed to yellow and the absorption band with maximum at 500 mfi was replaced by bands with maxima at 466, 437.5 and 350 m[mu]. The pink color was restored by oxygenation. These color changes were produced by all the substrates tested but not by related compounds that are not substrates. Evidence was obtained that the yellow products are emzyme-substrate complexes. 1,10-Phenanthroline, which inhibits the enzyme, prevents the formation of the yellow products and discharges their color. It is suggested that the yellow products are cu complexes. The inhibition of the formation of the complexes by 1, 10-phenanthroline is reversed by Ni2+ ions. The cu-free protein, obtained by incubating the diamine oxidase with sodium diethyldithiocarbamate, was catalytically inactive. The activity towards all the substrates tested was restored specifically by Cu2+ions. The Cu-free protein was orange-pink with maximum absorption at about 480 m[mu]. Substrate, added under anaerobic conditions discharged the color but yellow complexes were not formed. The pink color was not restored by oxygenation. When the cu-free protein was first reactivated by Cu2+ ions the addition of substrate produced the yellow complex and subsequent oxygenation restored the pink color. The yellow complexes were stable under anaerobic conditions but Cu2+ ions caused a rapid abnormal breakdown resulting in inactivation of the enzyme. This breakdown was prevented by ethylenediaminetetra-acetate.