sepharose and indoleacetamide

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.

Topics: Amidohydrolases; Ammonium Sulfate; Chromatography, Gel; Chromatography, Ion Exchange; Culture Techniques; Hydrogen-Ion Concentration; Indoleacetic Acids; Naphthaleneacetic Acids; Oryza; Sepharose; Substrate Specificity; Temperature