62 



CHAPTER IV 



PURIFICATION OF PROTEINS 

 FROM THE AROMATIC AMINO ACID PATHWAY 



This chapter includes the separation and purification 

 of the common pathway bifunctional DQT/SDH proteins from 

 Nicotiana silvestris suspension cells. Six to eight steps 

 of column chromatography, including a specific NADP* 

 affinity column have been used. Prephenate aminotransferase 

 and arogenate dehydrogenase were also purified by a series 

 of chromatographic steps . 



Results 

 DQT/SDH purification 



In crude extracts, activities for SDH and DQT (specific 

 activity of 61 and 13, respectively) were detected by 

 fluorometric or spectrophotometric assays. The enzyme (s) 

 are not stable at 4°C after several days of storage, unless 

 glycerol and DTT are present. Under these conditions 

 activities remained stable to freeze/thaw, at either -20 or 

 -70°C. Two peaks of activity for the bifunctional S-protein 

 were eluted from a Celite 545 column with an ammonium 

 sulfate gradient (Fig. 4-1) . Elution of the major peak 

 began at about 50% of ammonium sulfate saturation and was 



