68 



specific 2'5'-Sepharose-4B affinity column at 1 fiM NADP* and 

 18 mM NaCl (Fig. 4-3) . Activities for DQT and SDH remained 

 coincident for each protein. The fold purification at this 

 step was 195 for SP-I and 918 for SP-II. Only SP-I was 

 purified further by repeating the DEAE-cellulose 

 chromatography step (Epps-KOH buffer) , followed by a second 

 application on the affinity column. The final fold 

 purification was 1077 for the SP-I. Purification yields, 

 purities, specific activities and ratios of SDH/DQT 

 activities are shown in Table 4-1 and in Table 4-2. 



Throughout the purification of SP-I and SP-II, the 

 ratio of shikimate dehydrogenase to dehydroquinase activity 

 was calculated to be within the range of 4.7 to 6.2 for SP-I 

 (at an average of about 5.1) and within a range of 4.4 to 

 5.4 for SP-II (at an average of about 4.8) . Each protein was 

 concentrated on a PM-10 membrane to about 1 or 2 ml before 

 storage at -70° C. 

 Purification of ADH and PAT 



When ADH was purified along with the SP-II, it was realized 

 that they eluted with overlapping activity peaks on the 

 Celite 545 column using the decreasing ammonium sulfate 

 gradient at about 25 to 20% of saturation as shown in Fig. 

 4-4. A critical step of separation occurred when SDH did 

 not bind to HA and ADH bound to HA (Fig. 4-5) . Without the 

 inclusion of this step, the two enzymes co-purified 

 throughout the entire purification procedure, both eluting 



