202 GIOVANNI FELICE AZZONE 



property of being hydrolyzed by DNP. On the other hand, this result is 

 consistent with those hypotheses in which a reduced phosphorylated 

 electron carrier of the XH ~ P type is formed at the expense of ATP in 

 the presence of uncoupling agents. 



Therefore, it appears likely that once the reduced phosphorylated 

 intermediate is formed, it can either transfer electrons to the cytochrome 

 system by which ATP is regenerated, or to DPN in which case reduced 

 pyridine nucleotide is formed and inorganic phosphate is liberated. 

 Further work is in progress in order to define more precisely the reactions 

 by which the oxidation of succinate by the terminal respiratory chain 

 requires a supply of high energy phosphate. 



References 



1. Chance, B., and Hollunger, G., Fed. Proc. l6, 163 (1957). 



2. Chance, B., and Hollunger, G., Nature, Loud. 185, 666 (1960). 



3. Klingenberg, M., and Slenczka, W., Biochem. Z. 331, 331 (1959). 



4. Klingenberg, M., Slenczka, W., and Ritt, E., Biochem. Z. 332, 47 (1959). 



5. Azzone, G. F., and Ernster, L., Nature, Loud. 187, 65 (i960) 



6. Azzone, G. F., and Ernster, L.,_7. biol. Chem. 236, 1518 (1961) 



7. Azzone, G. F., and Ernster, L.,^. biol. Chem. 236, 15 10 (1961) 



8. Pardee, A. B., and Potter, V. R.,y. biol. Chem. 176, 1085 (1948). 



9. Tyler, D. B., J. biol. Chem. 216, 395 (1955). 



10. Singer, T. P., and Kearney, E. B., Arch. Biochem. Biophys. 61, 397 (1956). 



11. Azzone, G. F., Ernster L., and Klingenberg, M., Nature, Lond. 188, 552 

 (i960) 



12. Azzone, G. F., and Ernster, \^.,y. biol. Chem. 236, 1501 (1961) 



Discussion 



Chance: I think Dr. Azzone's paper has a great deal of information in it, it 

 takes time to digest. His remark about the non-phosphorylating preparation 

 oxidizing succinate directly is surely one that we must not forget in postulating 

 mandatory succinate oxidation, and I guess that it is one thing that just isn't 

 explained yet by your mechanism. We have the feeling that the ATP requirement 

 for succinate oxidation and ATP requirement for reduction of pyridine nucleotide 

 may not be identical in detail although they appear to be identical in the kind of 

 experiments that you have been doing. I think that the amytal sensitivity that 

 Dr. Ernster has already referred to gives a hint that this process does involve 

 carriers of the respiratory chain. I don't quite understand the basis on which 

 you conclude the uncoupling agents don't impair the formation of DPNH, or 

 maybe you meant they didn't impair the reactivation of succinate oxidation, 

 because I believe it is clear from our experiments that the DPNH formation is 

 highly sensitive to uncoupling agents. 



Azzone: Well, I think we must make a distinction here. If you mean that in 

 State 3, DPNH gets easily oxidized, then I agree with you that addition of dinitro- 

 phenol, which induces a State 3 condition, also makes it more difficult to observe 

 DPN reduction. But our conclusion that addition of uncoupling agents does not 



