Diamine oxidase of Lathyrus sativus seedlings. Purification and properties

Abstract

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings of Lathyrus sativus by MnCl₂ treatment, (NH₄)₂SO₄ and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had an M_r of 148,000 (6.46S) and was a dimer with similar sub-units (M_r 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the K_m for benzyl amine without any change in the Vmax Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to the L. sativus enzyme.