Discussion 147 



azide which could be quite clearly separated from one another (Myers, 

 D. K., and Slater, E. C. (1957). Biochem. J., 67, 572). Azide acts like a 

 dinitrophenol in that it stimulates the ATPase of freshly prepared liver 

 mitochondria, but also inhibits the DPN-stimulated ATPase or, as you 

 have just said, the magnesium-stimulated ATPase of aged mitochondria. 

 We were able to separate these two effects by a time sequence. The 

 stimulatory effect of azide is a rapid reaction and the inhibitory effect 

 is a slower reaction. You are studying the inhibitory action of azide 

 which is the slow reaction. Have you come across a stimulatory 

 effect? 



Lehninger : In intact mitochondria we were fully able to confirm this 

 two-phase effect which Boyer showed, but not with the digitonin 

 fragments — there we got only the one effect (Wadkins and Lehninger, 

 1959, loc. cit.) There does not seem to be biphasic action. I have 

 always been afraid that the ATPase activity of intact mitochondria is 

 much more complex than it is in digitonin fragments. I think there are 

 factors of membrane permeability, compartmentation and so on, which 

 tend to muddle up the issues. 



Lipmann : You showed that arsenate competes with phosphate. Does 

 DNP compete with the carrier? We have thought of this because of the 

 competitive inhibition of menadione reductase. Both dicoumarol and 

 DNP inhibit competitively this reductase in liver extracts. We thought 

 that might be the explanation. 



Lehninger: I do not think that we have had an appropriate experi- 

 mental situation to test such a competition. 



Racker : It has been found that other flavoproteins, such as the 

 quinone reductase, are inhibited by dinitrophenol (Wosilait, W. D., 

 Nason, A., and Terrell, A. J. (1954). J. biol. Chem., 206, 271). I wonder 

 whether this may be a rather non-specific effect and not necessarily 

 related to oxidative phosphorylation. 



Chance: It is apparent from Prof. Lehninger's mechanism that if 

 azide blocks equation 2 in phosphorylation, then you should have 

 dicoumarol stimulation of respiration above that obtainable by ADP 

 in your scheme (equation 3). I ask this because our kinetic data (Chance 

 and Williams, 1956, loc. cit.) show a rate-limiting step in the ADP 

 reaction which should be by-passed with dicoumarol or DNP. Have 

 you done this? 



Lehninger: Yes. The work with azide on a respiring system is, of 

 course, difficult because there can be some overlap of those concentra- 

 tions which uncouple and those which inhibit respiration. But within 

 the limits of this kind of experiment we can do that very nicely. We can 

 put in azide and on top of this get stimulation of respiration by DNP. 



Chance: That is above or equal to tlhe level you have obtained with 

 ADP only? 



Lehninger : Yes, about equal, we have never been able to get it beyond 

 that. 



Chance: Was this with digitonin particles or intact mitochondria? 



Lehninger: Digitonin particles. 



Chance: I do not believe that this is the case with the azide-treated 



