20 

 Preparation of N. silvestris extract 



Crude extracts for assay. Frozen, ground N. silvestris 

 cells were suspended (1:1) in 100 mM KPO4 buffer at pH 7.3 

 containing 20% glycerol, 1.0 mW phenylmethylsulfonyl 

 fluoride and 1.0 mM dithiothreitol, centrifuged at 35,000 x 

 g at 4°C for 20 min. Extracts were treated to 80% of 

 saturation with ammonium sulfate, centrifuged as above, 

 followed by resuspension of pellets in EPPS-KOH buffer (pH 

 8.6) with all of the above components and dialyzed with four 

 changes of buffer until ammonia could not be detected by 

 HPLC. Extracts not treated with ammonium sulfate were 

 desalted using PDIO columns in Epps-KOH buffer as above or 

 dialyzed overnight with 4 changes of the Epps-KOH buffer. 



Extracts for purification of ADH and PAT. Five-day N. 

 silvestris suspension cells were used in the purification of 

 ADH and PAT. Crude extracts were prepared as described 

 above and also included 0.1 mAT pyridoxal 5' phosphate and 

 1.0 mAf EDTA in order to stabilize PAT (5) . 



Extracts for S-protein purification. For purification 

 of the S-proteins, frozen, ground seven-day N. silvestris 

 suspension cells (300 g) were suspended into 300 ml of 100 

 mM KPO4 buffer, pH 7.8, containing 20% glycerol, 1 . mM DTT, 

 1.0 mM benzamidine, . 2 mM PMSF and 0.05 mM leupeptin. A 

 sample was immediately desalted using a PDIO column. 



