230 MARTIN KLINGENBERG 



nucleotide system, but also to be related to the reducing qualities of suc- 

 cinate or glycerol phosphate. This is supported by measurements of the 

 absorption of mitochondrial suspensions at the flavin nucleotide wave- 

 length, from which the amount of flavin nucleotide reduced has been 

 estimated. With succinate or glycerolphosphate several times more flavin 

 nucleotide is reduced than with pyruvate plus malate. Thus under these 

 conditions of optimum intramitochondrial energy supply, a relation of the 

 redox state between the pyridine nucleotide and the flavin nucleotide 

 systems appears to exist. 



When electron transport, and thus energy supply for oxidative phos- 

 phorylation, are inhibited by antimycin A, the adenine nucleotides are 

 phosphorylated to a low degree only. It is to be noted that under these 

 conditions the reduction of DPN and flavoprotein in the presence of 

 succinate or glycerolphosphate is also diminished. This indicates that not 

 only the reduction of DPN, but also of a part of flavoprotein is dependent 

 on functioning oxidative phosphorylation. Again, the redox states of the 

 DPN and flavin nucleotides behave in a parallel manner. 



An energy-dependent reduction of flavoprotein had been observed and 

 reported previously in flight muscle mitochondria [13, 14]. It v^as con- 

 cluded that this flavoprotein cannot be on the main pathway of the oxida- 

 tion of glycerolphosphate, since it was not reducible when electron 

 transport was inhibited by anaerobiosis or antimycin A. Thus this flavo- 

 protein may be reduced by the substrates through an energy-dependent 

 hydrogen transfer in a way similar to the mitochondrial DPN. 



Influence of exogenous ATP 



EFFECT OF ATP ON PYRIDINE AND FLAVIN NUCLEOTIDES 



In contrast to heart muscle and flight muscle mitochondria, the pyridine 

 and flavin nucleotides in isolated skeletal muscle mitochondria remain 

 oxidized if succinate or glycerolphosphate is added, although both sub- 

 strates are active hydrogen donors for these mitochondria [4, 15]. Only the 

 subsequent addition of ATP results in a large reduction of the DPN, as 

 shown in Fig. 2. On addition of phosphate, DPN becomes largely re- 

 oxidized. A similar ATP efl^ect can be obtained in heart muscle mito- 

 chondria with another type of flavin specific substrate, in the presence of 

 capronate. The extent of the DPN reduction is quantitatively measured by 

 enzymic pyridine nucleotide analysis (Table II). DPN is further reduced 

 to 45% by the addition of ATP in the presence of either glycerolphosphate 

 or succinate. With the DPN specific substrates, pyruvate plus malate, a 

 dift'erent picture emerges. In this case, DPN is reduced to about 32*^0 by 

 the substrates alone. ATP addition does not increase further the reduction 

 of DPN. 



