Xanthine dehydrogenase of pea seedlings: a member of the plant molybdenum oxidoreductase family

Xanthine dehydrogenase (XDH, EC 1.1.1.204) was purified to homogeneity from etiolated pea (Pisum sativum conv. speciosum) seedlings. The procedure involved initial purification with precipitants followed by two low pressure chromatographic steps. The partially purified enzyme was further subjected to FPLC on Superdex and Uno Q columns and to affinity-interaction chromatography on Affi-Gel Blue. Purity of the final enzyme preparation was checked by SDS-PAGE. Pea XDH forms a dimer of 2 × 150 kDa in the native state and is an acidic protein with pI 5.3. The enzyme shows quite stringent substrate specificity; only xanthine and hypoxantine are oxidized at a high reaction rate, some aldehydes such as indole-3-acetaldehyde are converted as well, but at rates lower than 3%. The enzyme was strongly inhibited by allopurinol, a typical inhibitor of molybdenum cofactor-containing enzymes, and less strongly by adenine and some cytokinins with aromatic side chain. N-terminal amino acid sequence of the pea XDH shows a high degree of homology to that of aldehyde oxidase (EC 1.2.3.1) from maize, a member of plant molybdenum cofactor enzymes and also to some other enzymes of this family.