TRYPTOPHAN OXIDIZING ENZYME AND BASIC PEROXIDASE ISOENZYMES IN ARABIDOPSIS-THALIANA (L) HEYNH - ARE THEY IDENTICAL

The in vitro conversion of [H-3]tryptophan by a plasma membrane enriched fraction from Arabidopsis thaliana (L.) Heynh. seedlings, grown in liquid culture, revealed indole-3-acetaldoxime (IAOX) as the only detectable reaction product. The pH optimum of the reaction was at pH 8, the K(m) value for tryptophan 12 muM. The formation of IAOX was stimulated about 10-fold by H2O2. Incubation experiments with solubilized proteins and membrane vesicles showed that the investigated enzyme(s) were bound covalent to the plasma membrane. Tryptophan oxidizing enzyme (TrpOxE) and peroxidase activity were not only found in the plasma membrane, but also in the culture medium. Specific IAOX forming activity was 74-fold and 6-fold higher compared to the crude extract and the plasma membrane fraction, respectively. After isoelectric focusing of solubilized plasma membrane and precipitated medium proteins, TrpOxE activity co-migrated with two prominent high pI peroxidase bands stained with benzidine-guaiacol. The zones of the IEF gel with peroxidase and TrpOxE activity were analyzed by SDS PAGE and revealed in all fractions a main protein band of ca. 55 kDa. TrpOxE activity and peroxidase activity were both inhibited by antisera directed against tobacco and horseradish peroxidase. TrpOxE activity and peroxidase activity were determined during plant development. TrpOxE activity peaked after 8 and 42 days, whereas peroxidase activity was consistently present during the whole life cycle. The inhibitory effects of indole derivatives, especially indole-3-glyoxylic acid, on (i) seedling development and (ii) on TrpOxE and peroxidase activity were also compared.