Isolation and properties of the enzyme system forming indoleacetic Acid.

The enzymatic conversion of tryptophan to ind,oleacetic acid (IAA) has been demonstrated in lower and higher plants. In some plants the pathway muist be limited to that involving ini(olepyrtuvic acid (5 ) while in others an alterniative pathway involving tryptamine also exists (6, Q). Alth,ouigh the enzyme system from the pineapple leaf converting tryptophlan and tryptamine to auixin was obtained by precipitation with ammonium stilfate (1), subsequent studies have used the supernatant fraction following centrifugation as the enzyme preparation. Suich a preparation has been fouind inadequate for the precise deterfmination of pathway and the characteristics of the enzymes involved. In the investigatio,n reported here a partial purification of the enzyme system from Avenia tissue has been achieved and some of its properties have been examined. Oats (Avenai satizva cv Victory) are grown in the dark at 240 and 82 % relative humidity for 86 to 9( hoturs. Five grams of frozen apical segments (1 cm long) of the coleoptiles are macerated in 50 ml of cold buffer (KH.PO4-Na,HPO4, 0.05 M, pH 7). The homogenate is centrifuiged f,or 40 minuites at 10,000 X g at 00. The 50 ml of stupernatant then is passecl throuigh a column of Sephadex G-25 (Pharmnacia, Uppsala), which is eqtuilibrated with the buffer, to remove compouinds with moleculair weights belowv 5000. The filitrate is saturated with ammonitim suilfate and flocculationis allowed to proceed for 16 houirs at 00. The residue is separated by cen:trifugation at 10,000 X g for 30 minuites at 0°. The resulting pellet is taken ilp in 50 ml of buffer and dialyzed against diluited bu,ffer (1 liter, 0.025 M) for 18 hours at 0°. The dialysate is passed throtigh a column of Sephadex G-200 (Phar,mra,cia, Uppsala) which is equilibrated with the 0.05 mI buffer to remove all molecules with molecular weights below 200,000. The filtrate with voltume of 50 ml is used as the enzyme preparation. Throughiotut the procedure precautions are taken to