In vitro and In vivo Inhibition of Plant Polyamine Oxidase Activity by Polyamine Analogues

MAIALE, SANTIAGO J; MARINA, M; SANCHEZ, D H; PIECKENSTAIN, F L; RUIZ, O A

PHYTOCHEMISTRY

Elsevier

Año: 2008 vol. 69 p. 2552 - 2558

0031-9422

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 affinity for spermidine and spermine (Km ~5.5 and 1.2 mM, respectively), and also oxidized norspermidine (Km ~64.0 mM). Natural and synthetic diamines, cyclohexilamine, the putrescine analogue 1-aminooxy-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 tetramine 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. 10 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 (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations 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 new insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.