Discussion 85 



taining DPN, rather than the synthesis of DPN from substances of 

 smaller molecular weight. 



Hess : How much ADP do you need? 



Slater : If ADP alone is added to mitochondria, there is half maximal 

 appearance of extra DPN (a sort of iiC^) at 200 (xm (2 x 10-* m), 

 whereas it occurs at 70 [xm if ADP is added in presence of 2 mM inorganic 

 phosphate. 



Lynen: Prof. Slater, you talk about concentrations of ADP and 

 phosphate added, but what Dr. Hess wants to know is whether you can 

 titrate the DPN '-^I coinpound. If your equations are correct, then you 

 would have to add to 1 [xmole of DPN'~-'I, 1 [jimole of ADP and inorganic 

 phosphate. 



Slater: We have not yet tried to titrate DPN~I with ADP. Its 

 average concentration, determined from the amount of extra DPN 

 formed on the addition of excess ADP, is 1-67 [j,moles/g. of protein. 

 That comes out within 10 per cent or so of the value reported from 

 Prof. Chance's laboratory (Eisenhardt and Schrachinger, 1958, loc. cit.), 

 which was determined from the amount of ATP which is rapidly 

 synthesized on the addition of ADP, which is really a titration of mito- 

 chondria for its '--' content. 



Chance: Prof. Slater has interpreted his experimental results on the 

 assumption that a compound of oxidized DPN is converted, in the 

 presence of ADP and inorganic phosphate, into free DPN which can 

 then be assayed by the alcohol dehydrogenase. 



ADP, Pj 



(1) DPN~I > DPN + ATP + X + I 



X 



Our interpretation of spectroscopic studies of intramitochondrial 

 pyridine nucleotide suggests that a DPNH compound (DPNH'^I) is 

 converted to DPN according to the following equations, which express 

 what we term the state 4-to-3 transition or the. quiescent-active state 

 transition : 



ADP, Pi 



(2) DPNH^I > DPNH + ATP + X + I 



(3) DPNH + rfp + H + > f p + DPN + 



ADP, Pi 



(4) DPNH^I + rfp + H + > DPN + + f p + ATP + X + I 



X 



The experimental data presented by Prof. Slater are consistent with 

 equations 2, 3 and 4: addition of ADP produces DPN + ; addition of 

 dinitrophenol produces DPN ; and addition of am>i;al diminishes DPN + 

 by blocking equation 3. The spectrophotometric traces indicate that 

 the "new form" of pyridine nucleotide is reduced, not oxidized. How- 

 ever, Prof. Slater's suggestion that the opposite conclusion is true raises 



