29 

 alcohol dehydrogenase (150,000 D) , BSA (66,000 D) and 

 carbonic anhydrase (29,000 D) plus sucrose were applied to 

 the column. 



Purification of pathway proteins 

 Purification of the S-proteins 



Crude extract of 54 ml was brought to 60% of ammonium 

 sulfate saturation at 4°C while stirring and was used for 

 the purification of the S-proteins. 



Celite 545 chromatography. After stirring the 60% of 

 ammonium sulfate saturation slurry for 3 min, 70 g of 

 Celite 545 was added, with continued stirring for about 20 

 min. The slurry containing 605 mg protein was applied to a 

 column and the eluate collected into a large cylinder. The 

 column volume of 174 ml was washed with 60% of (NH4)2S04 

 saturation-Epps-KOH buffer containing 20% glycerol and 1.0 

 mM DTT at pH 8.6 and was collected in a separate cylinder. 

 No activity was found in these eluates, although the protein 

 level was very high. A 500-ml gradient from 60 to 40% of 

 (NH4)2S04 saturation in the same buffer was applied and 8-ml 

 fractions were collected. Next a 40% of (NH4)2S04 

 saturation-Epps-KOH buffer of about 130 ml was applied. A 

 second 400 ml gradient of 40 to 0% of (NH4)2S04 saturation- 

 Epps-KOH buffer was applied, followed by a wash of buffer 

 without (NH4)2S04. Two peaks of SDH/DQT activities were 

 located, pooled individually, and dialyzed against the Epps 



