122 BRITTON CHANCE 



(0-28 compared to 0-30). As in the case of liver mitochondria, reduced 

 pyridine nucleotide shows a typical cycle of oxidation and reduction upon 

 addition of ADP. But the initial rate of DPNH oxidation is small compared 

 with the steady-state rate of oxygen utilization (o • 56 compared with 2 -o in 

 2-electron equivalents). Thus the significant features of this reaction are 

 the relatively slow changes in pyridine-nucleotide reduction states, which 

 lead nevertheless to very large magnitudes of changes in steady state. 



4mM 

 succinote 



State 4 

 (qlutamate) 



Spectrophotometric trace 



ccinate) 



Platinum 

 microelectrode trace "* 



340-374m^-j- 

 log Io/I = 0- 



h- 50 sec H 



Fig. 3. Illustrating increase of pyridine-nucleotide reduction caused by 

 adding succinate to glutamate-treated guinea-pig-kidney mitochondria. Down- 

 ward deflection of trace indicates increased absorbancy at 340 m/x measured with 

 respect to 374 m/it. Rates of pyridine-nucleotide reduction and oxygen utilization 

 indicated in jumoles/l./sec. Mitochondria diluted in sucrose-phosphate medium to 

 concentration of o-6 mg. protein/ml., pH 7-4, temperature 2$'- (Expt. 683-1). 

 (Reproduced with permission of The Journal of Biological Chemistry.) 



While the above experiments showed the important role of succinate 

 in activating DPN reduction in mitochondria they did not clearly rule out 

 the possibility that addition of succinate increased the concentration of a 

 DPN-linked substrate, for example malate, according to the sequence 

 of the citric acid cycle (Fig. 4). This hypothesis is largely ruled out in 



Succinate — ^ fumarate — ^ malate 

 Fig. 4. 



oxalacetate 



experiments illustrating the abrupt inhibitory effect of malonate upon 

 succinate-linked DPN reduction. For instance in experiments such as 

 those recorded in Figs. 2 and 3 addition of malonate causes an abrupt 

 decrease of pyridine-nucleotide reduction to the level previously obtained 

 in the presence of the DPN-linked substrate only. That this inhibition 

 occurs with no measurable induction period, as would have been expected 

 for a mechanism which depended upon accumulation of DPN-linked 

 substrate, rules against the simple hypothesis outlined in Fig. 4. 



