In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues.
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 (K(m) approximately 5.5 and 1.2 microM, respectively), and also oxidized norspermidine (K(m) approximately 64.0 microM). Natural and synthetic diamines, cyclohexylamine, 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 K(i) 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 K(i) values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K(i) 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 ( approximately 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.
Maiale
Santiago J
SJ
IIB-INTECH, Camino de Circunvalación de la Laguna km 6, Casilla de Correo 164, Provincia de Buenos Aires, B7130IWA Chascomús, Argentina.
Marina
María
M
Sánchez
Diego H
DH
Pieckenstain
Fernando L
FL
Ruiz
Oscar A
OA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001203:Ascomycota / Q000187:drug effects; D018554:Avena / Q000201:enzymology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D015394:Molecular Structure; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism*; D011437:Propylamines / Q000378:metabolism; D011549:Pseudomonas / Q000187:drug effects; D013312:Stress, Physiological; D013379:Substrate Specificity; D000097667:Polyamine Oxidase
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D006861:Hydrogen Peroxide; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011073:Polyamines; D011437:Propylamines; D000097667:Polyamine Oxidase; C046252:1-aminooxy-3-aminopropane