ASHIKAWA 



77 



were harvested and washed twice with sterile distilled water. Aliquots of cells 

 were then suspended in nitrogen-deficient medium (4 per cent dextrose : M/GO 

 KH 2 PO4:3/60 M NaHsPOi) and aerated for 48 to 11 hours at 30° C. During 

 this nitrogen starvation, the buffered dextrose medium in some of the cultures 

 was changed every 12 hours. At the end of starvation, the cells were centrifuged 

 and rewashed twice with sterile distilled water. Approximately 6 gram aliquots 

 of wet cells were suspended in 2-liter flasks containing 1500 ml YED (1:2 per 

 cent) and induced to grow aerobically at 30° for varying time intervals. The 

 growth curve of the cells and the corresponding bud counts for the first 26 

 hours are shown in figure 1. The yeast cells were then harvested at different 

 times, and microsomal particles were isolated according to Wolfe [23]. Cells 

 hand-ground with 100-grid Carborundum in mortar and pestle at 0° C were 

 extracted with several volumes of solvent (0.00125 M KH2PO4-K2HPO4 3:7, 



lOOrr 



\ 



CO 



_l 

 _l 

 UJ 



o 



r- 

 O 



"i r 



-o- 



A 



8 12 16 20 24 28 



HOURS AFTER INOCULATION 

 Fig. 1. Relationship of budding to growth of yeast cells, (a) Growth curve of 48-hour 

 nitrogen-starved yeast cells aerobically cultured at 30° C in YED (1:2 per cent), (b) Curve 

 correlating percentage of visible buds to corresponding growth stages. 



