Effect of 2,4-dichlorophenoxyacetic Acid on Proteolytic Activity of Red Kidney Bean Plants.

Recent work has slhown that treatment of plants with 2,4-dichlorophenoxyacetic acid (2,4-D) results in a reduction of carbohydrate and an accumulation of nitrogen (2, 10, 13, 14, 15). SELL et al. (14) and WELLER et al. (16) have observed differences in carbohydrate, nitrogen, and amino acid content that occur primarily in the proliferated stem tissue of the red kidney bean plants. LUECKE et al. (8) have also noticed changes in the content of several vitamins after treatment with 2,4-D. FELBER (3) and NEELY et al. (11, 12) have shown lower activity for peroxidase, alpha and beta amylase, and phosphorylase, and higher activity for pectin methoxylase in the treated red kidney bean plants. Suggestions have been made that the decrease in carbohydrate and the increase in amino acids and nitrogen content in the stem tissue were due to the conversion in part of the carbohydrates into proteins (14). If this postulate is valid then the proteolytic activity might be increased in the treated plants. This communication is a report of the results of an investigation on the effect of 2,4-D on the proteolytic activity of the red kidney bean plants. Sainples of leaf, stem, and root tissue of the red kidney bean plant, Phaseolus vulgaris, were prepared according to the procedure previously described by NEELY et al. (12). The proteolytic activity, using hemoglobin as the substrate, was determined on a sample of one gram of the powdered plant material by using MILLER'S (9) revision of the Ayre-Anderson method. Hemoglobin was employed as the substrate since it is a readily available protein and is soluble in solutions at a pH utilized in the analysis. The temperature of the medium was 480 C and a 0.1 M acetate buffer of pH 4.8 was utilized. The amino nitrogen was determined in a Van Slyke amino nitrogen apparatus. For the other proteolytic activity determinations, 0.750 gm. of the appropriate powdered plant material was suspended in 10 ml. of an aqueous 20% glycerol solution for four hours. At 30 minute intervals the reaction tubes were inverted several times to resuspend any settled material at the bottom of the tubes. After four hours the suspension was centrifuged, filtered, and the clear filtrate used as the enzyme preparation. Longer extraction periods did not increase enzymatic activity. The synthetic substrates consisted of 1% solutions of glycylglycine, L-cystinyldiglycine, L-cystinyldiglycyldiglycine and chloroacetyl-L-tyrosine. The glycylglycine and chloroacetyltyrosine were prepared according to