Polyamine oxidase was purified to homogeneity from leaves of water hyacinth by the criterion of sodium dodecyl sulfate gel electrophoresis (SDS disc PAGE). The enzyme showed a high specificity for spermidine and spermine (K. values 28 micromolar and 20 micromolar, respectively). The optimal pH of the enzyme for both spermidine and spermine was 6.5. The molecular weight of the enzyme estimated by Sephadex G-200 gel filtration was 87,000, while SDS disc PAGE pve a single band at the molecular weight of 60,000. Octamethylenediamine and quinacrine were strong inhibitors of the enzyme, but p-chloromercuribenzoate was without effect. A prosthetic group in the enzyme was identified as flavin adenine dinucleotide.PAOs' have been detected in several species of the Gramineae and characterized in oats (16,17), barley (15,16,18), corn (19,20), and millet (6). However, the PAO has not been found outside the Gramineae. In this paper, we describe the purification of PAO from water hyacinth, which does not belong to the Gramineae, and compare its properties with those of PAOs from other sources.MATERIALS AND METHODS Plant Material. Water hyacinth (Eichhornia crassipes Solms) was obtained from a local nursery. The harvested leaves were sterilized with 0.1% sodium hypochlorite for 10 min and washed thoroughly with deionized water.Preparation of Octamethylenediamine-Sepharose 4B. Octamethylenediamine was linked to CNBr-activated Sepharose 4B as described by Okada et al. (12).Enzyme Assay. Enzyme activity was assayed by measuring colorimetrically A-pyrroline, a product of the oxidation of spermidine, with o-aminobenzaldehyde as described earlier (19). The reaction mixture (3 ml), consisting of 150 ,umol K-phosphate (pH 6.5), 15 ,mol spermidine 3 HCI, 0.2 ml 0.2% o-aminobenzaldehyde (in ethanol), 250 ug beef liver crystalline catalase, and enzyme, was incubated at 300 C. The reaction was initiated by adding the enzyme and terminated by the addition of 0.2 ml 50% TCA. After centrifugation, the A at 435 nm was estimated. This method was used for the determination of the subcellular distribution, the purification, and the thermal stability of the enzyme. As an alternative assay, the enzyme activity was determined using a Clark oxygen electrode at 30°C. In this case, the reaction mixture (2 ml) contained 100 jAmol K-phosphate (pH 'Abbreviations: PAO, polyamine oxidase; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide. 6.5), 10 ,umol substrate, 250 ,ug beef liver crystalline catalase, and enzyme.One unit is defined as the amount of enzyme producing 1 jimol A-pyrroline or 0.5 Amol 02 uptake per min. Protein was determined by the procedure of Lowry et al. (9), using BSA as a standard.Subcellular Distribution. Leaves (30 g) of water hyacinth were homogenized in a chilled mortar with 60 ml of 50 mM Tris HCI buffer (pH 7) containing 0.45 M sucrose and 3 g of polyvinylpolypyrrolidone. The homogenate was passed through cheesecloth and then subjected to differential centrifugation.Purification of the Enzyme. Leaves of water h...