Fig 1.

Transformation of indoleacetate by cell extracts of Azoarcus evansii. IAA, indoleacetate; P1, enol form of 2-oxo-IAA; P2, keto form of 2-oxo-IAA; P3, unknown product. (A) Thin-layer chromatographic analysis. The assay was performed at 30°C with a total volume of 125 μl containing 0.1 M MOPS-K⁺ (pH 7.8), 0.2 mM [1-¹⁴C]IAA (2.75 kBq), 10 mM pyruvate, 2 mM NAD⁺, 6 U lactate dehydrogenase, and 75 μl cell extract (5.6 mg protein). Samples (24 μl) were taken at intervals, and the reaction was stopped by the addition of 30 μl of methanol to the mixture. (B) Time course of substrate consumption and formation of products. The assay was performed at 30°C with a total volume of 1.6 ml containing 0.1 M ammonium bicarbonate (pH 7.8), 4 mM IAA, 10 mM pyruvate, 2 mM NAD⁺, 60 U lactate dehydrogenase, and 0.96 ml cell extract (75 mg protein). Analysis was done by HPLC and UV detection at 260 nm. Note that the IAA concentration was 20-fold higher than that for panel A. IAA was quantified by UV detection at 260 nm using a standard. The amounts of the two main products that were combined in the graph were estimated by using the same absorption coefficient as that for IAA. This approximation is probably not exact and apparently causes a slightly larger amount of the main products formed than IAA consumed. (C) HPLC analysis and UV detection of the main products. Assay conditions were the same as those described above for panel B except that 146 kBq indole-[1-¹⁴C]acetate was added. Data for the 10-min sample (50 μl) are shown. HPLC system 2 was used. Note that in panels C and D, data for time zero to 22.5 min were omitted. (D) HPLC analysis and radiodetection of the main products. The sample, HPLC system, and assay conditions were the same as those described above for panel C. (E) HPLC analysis and radiodetection. Assay conditions were same as those described above for panel B except that 0.5 mM [phenyl-¹⁴C(U)]IAA (19 kBq) was added and the assay mixture volume was 0.5 ml. Data for the 30-min sample (70 μl) are shown. HPLC system 2 was used. (F) HPLC analysis and radiodetection. Assay conditions were same as those described above for panel B except that 0.5 mM [phenyl-¹⁴C(U)]IAA (20 kBq) was added; the assay mixture additionally contained 2 mM CoA, 2 mM ATP, and 5 mM MgCl₂; and the assay mixture volume was 0.75 ml. Data for the 30-min sample (70 μl) are shown. HPLC system 3 was used for a better separation of CoA thioesters.