32 

 Final steps of SP-I purification. SP-I was applied 

 once again to a DEAE column equilibrated in Epps-KOH buffer 

 as previously described. A 200-ml gradient from to 0.6 Af 

 KCl was applied. The SP-I activity peak fractions were 

 pooled and dialyzed against Epps buffer. An aliquot was 

 saved, and 83 ml containing 0.162 mg protein was applied to 

 the affinity column for the final step of purification. The 

 completed purification yielded 0.07 mg SP-I protein in 54 

 ml. 

 Purification of arogenate dehydrogenase 



ADH was purified by two methods: A) by a series of 

 steps of an established procedure which began with a to 

 45% ammonium sulfate fractionation and ended with the NADP* 

 affinity column (8) , or B) by the elution of ADH from a 

 Celite 545 column used to separate S-protein activities, 

 followed by HA column chromatography, DEAE column 

 chromatography, and the NADP* affinity column chromatography 

 described above . 

 Purification of prephenate aminotransferase 



PAT was purified by following an established procedure 

 (7) , which began with a 45 to 75% of ammonium sulfate 

 saturation precipitation step, followed by a 70°C treatment 

 to inactivate and then precipitate (by centrifugation) 

 contaminating aminotransferases, and ended with a specific 

 aminotransferase pyridoxamine phosphate (PMP) affinity 

 column . 



