144 

 RNA polymerase II that has a greatly reduced affinity for 

 a-amanitin (R. Bryant, personal communication, 1979). If 

 inhibition by ADGG-Con A is due to inhibition of polymerase, 

 then LAN-2 cells should be much less sensitive to ADGG-Con A 

 than the parent line A-9. 



Both A-9 and LAN-2 bind equivalent amounts of Con A 



125 



as determined from the binding of I-Con A (Figure 24) . 



LAN-2 cells were totally unaffected by either a-amanitin 

 or ADGG following 48 hours of exposure to concentrations 

 as high as 16 x 10 M. A-9 cells showed sensitivity to 

 both inhibitors and, as was found with CHO cells, the ADGG 

 was less toxic at equivalent doses than free a-amanitin 

 (Figure 28) . 



Following 15 minutes of exposure to Con A at a con- 

 centration of 40ng/ml, neither A-9 nor LAN-2 were inhibited 

 by more than 11%. ADGG at 2.5 x 10 M caused no inhibition 

 of LAN-2 and only slight inhibition of A-9 cells (13%) . 

 ADGG-Con A caused 40% inhibition of A-9 at concentrations 

 equivalent to those given for ADGG and Con A whereas the 

 LAN-2 cells were only inhibited to the extent observed from 

 Con A alone (Table 10) . This experiment provides conclusive 

 evidence in support of the proposed mechanism of toxicity 

 for ADGG-Con A. The Con A portion of the conjugate binds 

 to specific cell surface receptors followed by internali- 

 zation of at least the a-amanitin moiety which then inhi- 

 bits RNA polymerase II causing cessation of mRNA synthesis 

 and cell death. 



