214 E. C. SLATER, M. J. BAILIE AND J. BOUMAN 



addition of ADP are indeed due to changes in the oxidation-reduction 

 state of DPN, changes in TPN occurring more slowly. The finding of 

 TPNH in the mitochondria does, it is true, necessitate rather important 

 quantitative changes in the interpretation given by Chance and WiUiams, 

 e.g. whereas the latter concluded that transition from State 3 (active respira- 

 tion) to State 4 (respiration" inhibited "or controlled owing to lack of ADP) 

 was associated with a change in DPN from 50" „ to 99" ,, reduced, Klingen- 

 berg and Slenczka found that the corresponding values were 3"^ and 43% 

 reduced. Thus, even in the controlled state there was considerable DPN 

 in the oxidized form. 



Purvis (unpublished observations) has obtained similar results, which 

 are given in Fig. i. Addition of ADP and Pj to a mitochondrial suspension 

 causes oxidation of the reduced nucleotides due to exhaustion of endo- 

 genous substrate. After addition of substrate (/S-hydroxybutyrate) the 

 ADP is soon all phosphorylated, so that the mitochondria reach State 4, 

 in which about 5i"o of the total DPN (measured in State 2) and 6% of 

 the total TPN are in the oxidized form. Addition of more ADP brings the 

 mitochondria into State 3, in which 94'^',, of the DPN is oxidized. On the 

 other hand, there was little formation of TPN +. These results confirm 

 three of Klingenberg and Slenczka's findings, viz. (a) DPN is not com- 

 pletely reduced in State 4; (h) in both States 3 and 4 the predominant 

 reduced pyridine nucleotide is TPNH ; (c) DPNH responds to addition of 

 ADP much more rapidly than TPNH. As Klingenberg et. al. [9] have 

 pointed out, the presence of large amounts of DPN^ in the controlled 

 state removes one of the main arguments of Chance and Williams [7] 

 that DPNH is present in an inhibited form (DPNH~I). 



The high concentrations of reduced pyridine nucleotides in freshly 

 prepared liver mitochondria (cf. Table I) are presumably caused by the 

 presence of endogenous substrate and an "inhibited" respiration, owing to 

 the absence of ADP. The DPNH is only quite slowly oxidized when the 

 preparation is diluted in isotonic medium, or near isotonic (see Table HI — 

 cf. Kaufman and Kaplan [5]), but it is rapidly oxidized in hypotonic 

 medium without further addition, or in isotonic mediimi by addition of 

 dinitrophenol, 0-05 m P| or lower concentrations of P^ in the presence of 

 ADP (Table HI and Fig. i—cf. refs. [7, 17, 8, 11, 5]). 



Purvis' "extra DPN" 



One of the most important questions is whether the pyridine nucleotide 

 present in the mitochondria is free or bound. Huennekens and Green [2] 

 concluded that it was not free and that "the principal oxidases of the 

 cyclophorase complex occur as conjugated pyridinoprotein enzymes". 

 However, the concept that mitochondria were bound by a semi-permeable 



