The influence of growth substances upon sulphydryl compounds 



In this system acetate is transferred from acetyl phosphate to CoA enzymati- 

 cally, and the rate of this acetylation is proportional to the amount of 

 functional CoA present. A linear relationship between the disappearance 

 of acetyl phosphate and the quantity of CoA present is obtained from to 

 5xlO~^MCoA. Under the conditions utilized the acetyl phosphate 

 remaining after 1 5 minutes was measured by the ferric hydroxamic acid test. 



~ i^2x10''^W 



I 







5xio~^y\ 



5x10'^ 5x10''' 



Concn. of growth substance 



• Figure 10. Reaction of CoA with TIB A and 2-A-D. 



Final concentrations of constituents : 

 2-5 X 10-* M Co J 

 5X10-2M TRISpH 8 

 growth substances as indicated. 

 Volume : 0-2 ml. 

 Incubation time : 60 minutes. 

 Temperature: 30". 



Functional CoA remaining after incubation estimated according to Stadtman (1952), except that the 

 cysteine was deleted. 



The effects of TIBA and 2:4-D upon the reaction of CoA are shown in 

 Figure 10. It can be seen that while 2:4-D was only slightly inhibitory, TIBA 

 at 5 X 10^^ M completely destroyed the effectiveness of CoA in this reaction. 

 There is approximately one hundred times as much of the growth substance 

 as CoA present, but the time of incubation of the growth substance with CoA 

 was only one hour. An increasing effectiveness of the growth substance TIBA 

 with increasing time of incubation can be seen in Figure 11. It is strongly 

 suggested from these data that TIBA may interfere with the acyl transfer 

 functions of CoA through reaction with the sulphydryl group of the coenzyme. 



Earlier work on sulphydryl inactivating agents has frequently shown a 

 reversal of the inactivation with cysteine. Dickens (1933) reports such a 

 reversal of iodoacetic acid inhibition of rat liver glyoxalase and suggests from 

 his data that the iodoacetate attacks a prosthetic group rather than the 

 enzyme itself. We were able to obtain a partial reversal of the inaction of 

 CoA by TIBA as shown in Figure 12. This reversal, however, represents only 

 a small fraction of the TIBA inhibition. In the case of 2:4-D, however, 

 cysteine comxpletely reversed the inhibition obtained. It is possible that 



279 



