Enzymatic Dehydration of 3 - Hydroxymethyloxindole Received for publication March

Abstract

Crude and partially purified extracts of wheat (Triticum vulgare, red variety) germ catalyze the dehydration of 3-hydroxymethyloxindole to 3-methyleneoxindole. Examination of the ultraviolet absorption spectrum of a reaction mixture consisting of either the extract or partially purified enzyme and 3hydroxymethyloxindole, shows that this oxindole has undergone complete dehydration to 3-methyleneoxindole. TPNH-linked 3methyleneoxindole reductase, also a constituent of the wheat germ extract, can be separated from the dehydrase by passage through an Agarose 15 column. Utilizing these partially purified enzymes, it can be demonstrated that the dehydrase activity found in wheat germ is a discrete enzymatic function. The pertinence of the oxindole pathway of LAA metabolism to higher plants is suggested by the finding that intact pea seedlings as well as their extracts can oxidize IAA to HMO' and reduce its dehydration product, MeOx, to 3-methyloxindole (7). A highly purified MeOx reductase from peas showed complete substrate specificity for MeOx. Furthermore, MeOx reductase from peas exists in multiple forms, with differential sensitivity to the synthetic auxins, 2,4-D, and naphthaleneacetic acid (5). Demonstration of the enzymatic oxidation of IAA to HMO and the reduction of MeOx by a highly purified plant MeOx reductase for which MeOx is a specific substrate strongly suggest that the oxindole pathway functions in higher plants. The nonenzymatic dehydration of HMO can proceed at a measurable rate, especially under conditions of physiological pH and ionic strength (6, 7). An important gap in the evidence for the oxindole pathway of IAA metabolism has been the failure, until now, to demonstrate an enzymatic acceleration of the dehydration of HMO to MeOx. We present evidence that extracts prepared from wheat seedlings and wheat germ can catalyze this dehydration via HMO dehydrase. The current status of the oxindole pathway is described in Figure 1. MATERIALS AND METHODS Chemicals. 3-Bromooxindole-3-acetic acid was prepared by reaction of IAA with N-bromosuccinimide (2). This compound on solution in water is rapidly converted to MeOx. The latter was purified by chromatography with 5% isopropanol in water (1). HMO was prepared by photooxidation of IAA in 'Abbreviations: MeOx: 3-methyleneoxindole; HMO: 3-hydroxymethyloxindole. the presence of riboflavin (1). Other chemicals were of reagent grade. Plant Material: Treatment and Fractionation. Wheat seeds (Triticum vulgare, red variety) were rinsed and soaked in distilled water for 16 hr at room temperature. The seeds were covered with moistened vermiculite and incubated at 23 C for 7 days in total darkness. Seedlings (100 g) were rinsed for 1 hr with tap water and homogenized in a chilled blender with 100 ml of 20 mM potassium phosphate, pH 7.0. Subsequent manipulations were carried out at 4 C. The homogenate was filtered through eight layers of cheesecloth and centrifuged at 37,000g for 15 min. The clarified extract was dialyzed overnight against 100 volumes of the same buffer. Wheat germ, obtained from Nutritional Biochemicals Corporation, was defatted using a previously described method for pea flour (4). One hundred grams of defatted wheat germ were extracted with 350 ml of 20 mm potassium phosphate, pH 7.0, and centrifuged at 37,000g for 15 min. The supernatant was then centrifuged for 90 min at 176,000g. A portion of the clear extract was dialyzed as above, and the remaining portion was made 1% with respect to streptomycin sulfate. The precipitated nucleic acids were removed by centrifugation. The extract was dialyzed as before, and brought to 40% of saturation with ammonium sulfate by addition of solid ammonium sulfate. After 1 hr the precipitate was removed by centrifugation. The ammonium sulfate concentration was then increased to 60% of saturation. After 1 hr, the resultant precipitate was collected by centrifugation, dissolved in a minimal amount of 20 mm potassium phosphate, pH 7.0, and dialyzed against the same buffer for 18 hr. This fraction (500 mg protein) was applied to a column (4.9 cm2 X 85 cm) of Agarose 15 equilibrated with 20 mm potassium phosphate, pH 7.0. The column was eluted with one liter of the same buffer. All the proteins containing HMO dehydrase and MeOx reductase activity were eluted within 750 ml. The fraction size was 10 ml. The levels of the two enzymes in each fraction were determined. The 100-fold purified MeOx reductase was prepared from pea flour (4). Analytical Procedures. To determine the concentration and extinction coefficient of HMO, a sample was allowed to undergo complete dehydration to MeOx during storage overnight at room temperature. The concentration of MeOx and therefore that of the original HMO was calculated from absorption at 248 nm using the published extinction coefficient of MeOx (3). Measurement of MeOx Reductase. MeOx reductase activity of the 176,000g supernatant and the fractions obtained after passage through the Agarose 15 column was measured by a previously described method (4) and expressed as the decrease in absorbance at 340 nm, due to oxidation of TPNH per milligram of protein per min. Measurement of HMO Dehydrase. HMO dehydrase was estimated by the increase in absorbance at 248 nm due to the formation of MeOx (6, 7). The optimal pH conditions for this

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@inproceedings{BasuEnzymaticDO, title={Enzymatic Dehydration of 3 - Hydroxymethyloxindole Received for publication March}, author={Pinaki Basu and Vinita Tuli} }