Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells
Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells
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DOI：
10.1007/s10265-004-0146-6
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发表时间：
2004-06-01
影响因子：
2.8
通讯作者：
Ikuta, A
中科院分区：
文献类型：
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作者：
Arai, Y;Kawaguchi, M;Ikuta, A
经凝胶过滤和阴离子交换柱层析，水稻细胞中吲哚-3-乙酰胺（IAM）水解酶的活性增加了约628倍，经凝胶过滤和十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计，JAM水解酶的分子量分别约为50.5 kD和50.0 kD。ne酶在pH 6.0 ~ 6.5时活性最高。这种酶在4到50度的热处理中是稳定的，在52度的热处理中效果最佳。在至少143天内，活动保持在4度。纯化后的酶段除能水解JAM及其同系物1-萘-乙酰胺外，还能水解吲哚乙酸乙酯（Et-IAA），但不能水解吲哚-3-乙腈。JAM和Et-IAA的Km值分别为0.96 mM和0.55 mM。虽然该酶的分子质量与参与肿瘤形成的农杆菌JAM水解酶非常相似，但其高Km值等生化特性与农杆菌JAM水解酶有很大差异。基于这些酶的性质，我们将讨论酰胺水解酶是否参与水稻细胞生长素的生物合成。
The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography The molecular masses of the JAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. ne enzyme exhibited maximum activity at pH 6.0-6.5. The enzyme was stable against heat treatments between 4 and 50degreesC and works optimally at 52degreesC. The activity remained constant at 4degreesC for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to JAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for JAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of JAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.