Control of Rate of Intracellular Respiration 75 



of DPN and DPNH by specific enzymic fluorometric methods 

 applied to acid and alkaline extracts of liver mitochondria 

 after different times of incubation at 30°, in the presence of 

 air, inorganic phosphate or ADP. After about 5 minutes' 

 incubation, all the DPNH initially present had been oxidized, 

 but the amount of DPN appearing greatly exceeded the 

 amount of DPNH oxidized (i.e. after 5 minutes, the DPN 

 content exceeded the amount of DPN + DPNH initially 

 present). The extra DPN must have been derived from some 

 precursor. DNP was found effective in place of ADP or in- 

 organic phosphate, but ATP was ineffective. This strongly 

 suggests that the precursor is connected with oxidative 

 phosphorylation and, in fact, it has the properties expected of 

 DPN '^' I^. Mean values ([jimoles/g. protein) for the concen- 

 trations of the various forms in rat liver mitochondria were : 

 DPN, 105; DPNH, 1-60; DPN -- I, 1-67. The value for 

 DPN '^ I agrees closely with the estimate by Eisenhardt and 

 Schrachinger (1958) of the amount of high-energy compounds 

 in liver mitochondria (1-5 [xmoles/g. protein) obtained by 

 determining the rapid increase in ATP which occurs im- 

 mediately after the addition of ADP to liver mitochondria. 



It should be emphasized that the proposed DPN z^- 1 has 

 not yet been directly identified in either the acid or alkaline 

 extracts of mitochondria*. Its existence is inferred from the 

 increased (DPN + DPNH) which is found after incubation 

 with ADP, inorganic phosphate, or DNP. 



The concentration of the supposed DPN ^-^ I in liver 

 mitochondria after various treatments was measured by 

 determining the difference between the total diphosphopyri- 

 dine nucleotide content (measured by incubating untreated 

 mitochondria at 30° with ADP or DNP, followed by deter- 

 mination of the DPN content) and the sum of the DPN and 

 DPNH contents measured on acid and alkaline extracts, 

 respectively, of the treated mitochondria. The concentration 



* Note added in proof. Dr. Purvis has now obtained evidence of a substance 

 in alkaline extracts of fresh mitochondria which yields DPN on the addition 

 of aged mitochondria to this neutralized extract. 



