Ill 



The media was removed followed by three washes of fresh 

 media which were pooled and counted for determination of 

 eluted H-DM-ADGG-Con A. Identical control cultures were 

 processed without exposure to media for determination of 

 the total amount of H-ADGG-Con A that bound to the cells 

 during 15 minutes. The cells exposed to media were pro- 

 cessed for determination of remaining cell associated activ- 

 ity. This experiment allowed for estimation of the amount 

 of conjugate that is removed by exposure to carbohydrates 

 and glycoprotein components of the serum present in tissue 

 culture media. Exposure to media resulted in 60% of the 

 total activity being released from the cells. The remaining 

 40% (4.2 x 10 molecules/cell) is irreversibly associated 

 with the cell and is presumably internalized. This fraction 

 of the conjugate would be expected to be that which causes 

 inhibition of cellular activity. 



The ability of Con A conjugates of a-amanitin to bind 

 to specific cell receptors via the Con A moiety has been 

 clearly demonstrated by the previous experiments. Whether 

 this binding would result in targeting of the a-amanitin 

 with subsequent enhanced toxicity of the conjugate in com- 

 parison to free a-amanitin was examined in the following 

 experiments . 



Twelve hour cultures of II— 7 CHO cells were exposed to 

 ADGG-Con A, Con A or free ADGG for 15 minutes at 22°C, 

 washed and incubated at 37 °C for an additional 48 hours as 

 described in Materials and Methods. Toxicity was 



