Arai Yoshitaka, Kawaguchi Masayoshi, Syono Kunihiko, Ikuta Akira
Department of Biological Science and Technology, Science University of Tokyo, Noda, Chiba, Japan.
J Plant Res. 2004 Jun;117(3):191-8. doi: 10.1007/s10265-004-0146-6. Epub 2004 Mar 23.
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 IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0-6.5. The enzyme was stable against heat treatments between 4 and 50 degrees C and works optimally at 52 degrees C. The activity remained constant at 4 degrees C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM 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 IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM 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.
通过凝胶过滤和阴离子交换柱色谱法，水稻细胞中吲哚 - 3 - 乙酰胺（IAM）水解酶的活性提高了约628倍。通过凝胶过滤和十二烷基硫酸钠聚丙烯酰胺凝胶电泳估计的IAM水解酶的分子量分别约为50.5 kD和50.0 kD。该酶在pH 6.0 - 6.5时表现出最大活性。该酶在4至50摄氏度的热处理下稳定，在52摄氏度时活性最佳。在4摄氏度下，活性至少143天保持恒定。纯化的酶组分除了能水解IAM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。该酶对IAM和Et - IAA的Km值分别为0.96 mM和0.55 mM。尽管该酶的分子量与参与肿瘤形成的根癌土壤杆菌的IAM水解酶非常相似，但其生化特性包括高Km值与根癌土壤杆菌的酶有很大不同。基于这些酶的特性，我们将讨论这种酰胺水解酶是否参与水稻细胞中生长素的生物合成。
