23 

 1 h. The supernatant s were desalted by passage through 

 Sephadex G-25 columns before fluorometric assay. 



Enzyme assays 

 Aminotransferases 



Prephenate aminotransferase. Prephenate 

 aminotransferase was usually incubated with concentrations 

 of 20 xnM L-glutamate and 0.8 mW prephenate (unless otherwise 

 stated) , incubated at 37°C for 10 min (or as otherwise 

 stated) and then assayed by HPLC as an orthopthalaldyhyde 

 derivative in a 60% methanol/40% of 20 mM KPO4 buffer 

 system. Peaks of PHE were quantified before and after 

 acidification of the product of the reaction, AGN, in IN HCl 

 for 10 min at 37°C. 



Aminotransferase couples. Other aminotransferase 

 assays included an amino acid donor (ASP, TYR, LEU or ALA) 

 and keto acid (PPY) at concentrations as stated and were 

 assayed similarly by HPLC, before and after acidification 

 when applicable. Controls included samples without amino 

 acid donor or keto acid. 

 Aroaenate dehydratase 



Arogenate dehydratase was assayed according to Jung et 

 al. (43) by measurement of OPA derivatives following HPLC 

 where the peak of AGN formed were monitored before and after 

 acidification to PHE. Concentration of . 5 mM AGN, 0.25 mAf 

 TYR (activator), 50 mM EPPS-KOH buffer at pH 8.6 and enzyme 



.J 



