21 

 equilibrated with 50 mAf EPPS-KOH buffer at pH 8,6, 

 containing the same components as above and labeled as crude 

 extract . 

 Preparation of cultured cells for assay of ADH levels 



The isogenic suspension cell -culture line of N. 

 silvestris was grown in continuous light with gentle shaking 

 at 150 rev/min. The growth properties of a N. silvestris 

 suspension cell line (ANS-1) in lag, exponential, and 

 stationary phases of growth have been characterized 

 throughout a conventional growth cycle (referred to as E 

 cells) and for cells which have been maintained in 

 exponential growth for more than 10 generations, EE cells 

 (10) . For routine subculture, 80 ml of suspension-culture 

 medium were inoculated with 20 ml of 7 -day stationary-phase 

 cells in 500-ml Erlenmeyer flasks. EE cells (2 g wet 

 weight) were transferred to fresh medium on day 7 when cells 

 reached about 1 X 10^ cells ml"^. Daily samples were 

 harvested using Miracloth for filtration, washed with 

 distilled water, ground and frozen in liquid nitrogen, and 

 stored at -70°C until extract preparation. Daily sampling 

 was also done to determine a cell count by mixing 1 ml of a 

 50 ml culture with 12% chromium trioxide (helps to separate 

 cell clumps and visualize cells more easily) . Samples were 

 heated to 70°C for 2 to 5 min before counting in a Fuchs- 

 Rosenthal counting chamber at 20x magnification. The 

 remaining 49 ml of culture were filtered and cells were 



■:-M 



.OSJ 



;^ 



