lAA OXIDASE INHIBITOR AND MORPHOGENESIS 143 



(Tang and Bonner, 1948). It was therefore simple to demonstrate 

 directly the presence and gradient of this substance inferred from the 

 concentration experiments of the section above. 



As before, 500-mg fr. wt. samples of the various tissues were har- 

 vested, ground to a brei, briefly centrifuged to remove debris, and 

 made up to a final volume of 50 ml in O.IM pH 6.1 phosphate buffer. 

 The entire volume was then enclosed in a cellophane dialysis tube, and 

 permitted to stand overnight at 2°C in contact with 500 ml of O.OIM 

 pH 6.1 phosphate buffer, in a glass cylinder. The resulting dialyzed 

 enzyme was then assayed for activity and was compared with a sample 

 of the fresh brei, and with a sample of brei stored in a dialysis tube 

 overnight at 2°C, but not in contact with dialysis fluid. The data for 

 terminal bud brei are shown in Table I. The other tissues yielded 

 similar results. 



Table I. Effect of Dialysis on Activity of lAA Oxidase Preparations from the 



Terminal Buds of 2-Week-Old Light-Grown Peas. (Concentration of brei was 20 mg 



fr. wt. equivalent per 10 ml reaction mixture. 10"* if MnClo, 10"* if DCP, and 



2 X 10"* M lAA added. Salkowski technique employed) 



Salkowski Colors after Minutes 



It is clear that the activity of the enzyme is increased by dialysis, as 

 previously reported for this enzyme (Galston and Baker, 1951). The 

 inference is that a dialyzable inhibitor has passed out of the bag into 

 the surrounding dialyzing fluid. 



To demonstrate the latter point more directly and to obtain quanti- 

 tative data on inhibitor content, the dialysis experiment was repeated 

 with 50-ml breis of various tissues (= 500 mg fr. wt.) being dialyzed 

 against 50 ml O.IM buffer. This time, the dialyzate was harvested, and 

 0.5 ml of the fluid added to 10 ml of reaction mixture containing, as 

 enzyme, 1 ml of inhibitor-free brei (= 100 mg fr. wt.) from the third 

 internode of 7-day-oId etiolated Alaska pea seedlings. The results are 

 shown in Table II. 



