Purification and Characteristics of Diamine Oxidase from the Apoplast of Pea Epicotyls

Diamine oxidase was solubilized by stirring with a buffer from the apoplast of pea epicotyls and purified to homogeneity by one−step technique. The specific activity of the purified diamine oxidase was22.5U/mg protein. SDS gel electrophoresis showed a single band at a molecular weight of70KDa(1%SDS and 5%mercaptoethanol, at100°C for10min)and main band at a molecular weight of140KDa(1% SDS only, at100°C for10min). The enzyme activity was inhibited strongly by carbonyl reagents but was little inhibited by chelaters. The effect of the copper addition was not admitted at all. Bulletin f Aichi Univ. of Education,58(Natur l Sciences), p.61―64, March,2009 Yoko SAKAKIBARA・ Hiroshi YANAGISAWA 62 ― ― Tris succinate pH8.0. The enzyme was concentrated with centrifuge dialysis method or in cellophane tube in contact with polyethylene glycol20,000, and was kept at4°C until use. (Step2) 2.4.SDS electrophoresis and enzyme staining SDS electrophoresis was performed as described by Laemmli. As standard protein markers, Mark11was used. (Tefco) Proteins were visualized with a silver staining kit. (Nacalai tesque) 3.Results and discussion 3.1.Enzyme purification The enzyme was purified by SP-Sepadex C-50. The results of purification are summarized in Table1. The yield of the enzyme was88.4%, the purification resulted in84-fold, and the specific activity of the enzyme was22.5U/mg protein, the specific activity of DAO that has been reported is69.6U/mg proteinand45.9U/mg protein, and the specific activity of this report is the lowest though the purified enzyme is a single protein(Fig. 1). 3.2.SDS electrophoresis To examine the purity and subunit structure of the enzyme, SDS electrophoresis was performed. SDS electrophoresis showed that the purified enzyme was single and the molecular weight of the subunits with1% SDS and5%2-mercaptoethanol treatment at100°C for10min is70KDa. And a molecular weight of main band with1% SDS treatment at100°C for10min is 140KDa (Fig. 1). The purified enzyme was a dimmer. These results agreed with other diamine oxidases. The molecular weight of the purified enzyme was different from the previous report though the origin was the same. Because DAO is known as a glycoprotein, glycans obtained by different purification methods might be different. Step Total activity Total protein Specific activity Purification Yield (Unit) (mg) (U/mg) (-fold) (%) 1.Apoplasts Fraction 45.8 171 0.268 1 100 2.eluate from SP-Sephadex C-50 40.5 1.8 22.5 84 88.4 Table1.Purification of diamine oxidase from the apoplast of pea epicotyls FIG.1.SDS electrophoresis of diamine oxidase Line1, apoplast fraction12.6μg; Line2, eluate from SP-Sephadex24.9μg(1%SDS at100°C for10min); Line3, eluate from SP-Sephade x24.9μg(1% SDS and5% mercaptoethanol at100°C for10min); Line4, standard protein markers. Purification and Characteristics of Diamine Oxidase from the Apoplast of Pea Epicotyls 63 ― ― 3.3.Inhibitions of carbonyl reagents and copper chelaters In general, diamine oxidase contains topaquinone (TPQ)and copper, the purified enzyme was strongly inhibited by phenyl hydrazine, semicarbazide, and hydroxylamine. It was suggested that the purified enzyme contained TPQ. And the purified enzyme was weakly inhibited by copper chelaters. (Table2)The purified enzyme might contain not copper but other metals. 3.4.Effect of copper addition The copper addition did not activate the enzyme activity. (Table3) It was suggested that the purified enzyme did not contain apo-enzyme (enzyme to lack copper).