76 E. C. Slater and W. C. Hulsmann 



of DPN '-^ I determined in this way increased by incubation 

 with ADP, nicotinamide and amytal (0-8 mn). Subsequent 

 dilution and addition of DNP caused the formation of more 

 DPN. 



These interconversions of the various forms of DPN can be 

 explained on the basis of reactions (l)-{3b), and (6), in which 

 AH2 is DPNH and B is fp. Addition of ADP or DNP liberates 

 I^ from its combination with X and promotes the oxidation of 

 DPNH by equation (1). The simultaneous liberation of X 

 promotes the formation of DPN from DPN '^ Ij. The 

 accumulation of DPN ^^ I^ in the presence of ADP and low 

 concentrations of amytal suggests that amytal acts on 

 reaction (2). It might be added that these explanations 

 require that the intermediate is DPN '^-' I, not DPNH --^^ I. 



The effects of addition of substrate (in the presence of 

 oxygen, but absence of phosphate acceptor) on the concentra- 

 tions of the three forms of diphosphopyridine nucleotide are 

 particularly interesting. The addition of a-ketoglutarate, 

 glutamate or succinate caused a decrease of DPN, while the 

 DPNH either decreased or did not change (see Table IV). In 

 other words, DPN '^ I increased. The effects of a-ketoglutar- 

 ate and glutamate are in agreement with expectations accord- 

 ing to the following sequence (where SH2 = substrate; S = 

 product) : 



DPN -- Ii + X ^ DPN + X ^ Ii (12) 



Table IV shows that g-hydroxybutyrate caused an increase 

 of both DPN and DPNH, with consequent decrease of DPN 

 '^ I. This is unexpected, but could be explained if this 

 substrate has a slight uncoupling activity. 



The decrease of DPNH found after the addition of succinate 

 is in contrast to the conclusion drawn by Chance (1956), on 

 the basis of direct spectrophotometric observations of liver 



