lAA OXIDASE INHIBITOR AND MORPHOGENESIS 141 



overnight at 2°C v^ an equal volume of 0.0 IM pH 6.1 phosphate 

 buffer, the dialyzate being used as the inhibitor, (b) Brei was immersed 

 in a boiling water bath for 5 min, and the resulting precipitate was re- 

 moved by centrifugation or filtration, and the supernatant liquid was 

 used as inhibitor, (c) Intact tissue was boiled in distilled water or in 

 dilute buffer (O.IM, pH 6.1), and the resulting liquid was used di- 

 rectly as the inhibitor. 



All assays for lAA oxidase activity were made by the use of the 

 Salkowski colorimetric technique (Tang and Bonner, 1947) readings 

 being made 20 min after mixture of solution and reagent. Ten milli- 

 liters reaction mixture containing 10 "^M MnCb and 2,4-dichloro- 

 phenol (DCP) (Goldacre et al, 1953), 2 X 10" W lAA, buffer and 

 enzyme were placed in 50-ml Erlenmeyer flasks shaken 96 cycles/min 

 at 30°C in an Aminco Dubnoff metaboHc shaking incubator. One- 

 milliliter aliquots were removed at intervals and pipetted vigorously 

 into 4 ml of Salkowski reagent, then stirred vigorously. The resulting 

 colors were read 20 min later on a Klett colorimeter equipped with a 

 No. 54 green filter. 



Protein nitrogen determinations were made as previously described 

 (Galston and Dalberg, 1954). 



The lAA was the Eastman product, and the gibberellic acid used in 

 certain experiments was kindly supplied by Dr. P. W. Brian of Imperial 

 Chemical Industries, England. 



Results 



Distribution of Enzymatic Activity in Alaska Pea Plants. Two- 

 week-old Alaska pea plants, grown at 17°C and 24 hr photoperiod, 

 were divided into terminal bud, successive internodes, and leaves. Five 

 hundred milligrams of each kind of tissue was then ground, made up 

 to 50 ml brei, and assayed for lAA oxidase activity at various levels 

 of tissue equivalent per 10 ml reaction mixture. The results are shown 

 in Fig. 1. 



The following conclusions may be drawn from these data: ( 1 ) lAA 

 oxidase activity is high in terminal bud and young stem tissues, but 

 falls rapidly as the concentrations of tissue per standard volume of 

 reaction mixture is raised. This is best interpreted in terms of a high 

 level of inhibitor present in the brei. (2) With the older stem tissues, 



