CONICET | Buscador de Institutos y Recursos Humanos

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affin- ity for spermidine and spermine (Km _ 5.5 and 1.2 lM, respectively), and also oxidized norspermidine (Km _ 64.0 lM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-amino-oxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a Ki value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetra- mine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15 triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with Ki values of 5.8, 110.0 and 7.6 nM, respectively, where CH4 was a weak competitive inhibitor with a Ki value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with con- centrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited (_85%) S. sclerotiorum mycelial growth on Czapek- Dox medium. Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.

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