Maiale Santiago J, Marina María, Sánchez Diego H, Pieckenstain Fernando L, Ruiz Oscar A
IIB-INTECH, Camino de Circunvalación de la Laguna km 6, Casilla de Correo 164, Provincia de Buenos Aires, B7130IWA Chascomús, Argentina.
Phytochemistry. 2008 Oct;69(14):2552-8. doi: 10.1016/j.phytochem.2008.07.003. Epub 2008 Sep 8.
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.
使用包括渗透洗涤技术在内的简单四步纯化方案，将燕麦（Avena sativa L. cv. Cristal）幼苗中的多胺氧化酶纯化至同质。该酶对亚精胺和精胺具有高亲和力（Km分别约为5.5和1.2 μM），并且还能氧化降精胺（Km约为64.0 μM）。天然和合成二胺、环己胺、腐胺类似物1-氨基氧基-3-氨基丙烷以及几种多胺类似物对多胺氧化酶活性具有抑制作用，且均不是底物。当添加反应产物1,3-二氨基丙烷时，未观察到对亚精胺氧化的抑制作用。相比之下，1-氨基氧基-3-氨基丙烷表现出混合抑制动力学，Ki值为0.113 mM。此外，体外酶活性测定表明，寡胺[3,8,13,18,23,28,33,38,43,48-十氮杂-（反式-25）-五十碳烯]、四胺1,14-双-[乙氨基]-5,10-二氮杂十四烷和五胺1,19-双-[乙氨基]-5,10,15-三氮杂十九烷对多胺氧化酶表现出强效竞争性抑制活性，Ki值分别为5.8、110.0和7.6 nM，其中环己胺是一种弱竞争性抑制剂，Ki值为0.5 mM。这些类似物在体外不抑制核盘菌（Sclerotinia sclerotiorum (Lib.) De Bary）和绿黄假单胞菌（Pseudomonas viridiflava (Burkholder) Dowson）的菌丝生长。相反，在与多胺类似物所用浓度相似的情况下，双胍辛胺（一种著名的杀菌剂，同时也是一种多胺氧化酶抑制剂）抑制了（约85%）核盘菌在查氏培养基上的菌丝生长。最后，类似物1,19-双乙氨基-5,10,15-三氮杂十九烷抑制了体内玉米叶片切段中观察到的多胺氧化酶活性。所得结果为研究多胺氧化酶活性对植物生物和非生物胁迫的影响提供了见解。
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