RE\^RSAL OF ELECTRON TRANSFER IN THE RESPIRATORY CHAIN 121 



were afforded by the inhibitory effects of amytal upon the rate of pyridine- 

 nucleotide reduction caused by succinate. Since amytal inhibits DPNH 

 oxidation by the mitochondrial respiratory chain [ii, iia], one would 

 have expected that the effectiveness of succinate in intercepting oxidizing 

 equivalents would have been even greater in the presence of this inhibitor. 

 On the other hand, the observation of Klingenberg et al. [5] that a-gly- 

 cerophosphate causes enhanced pyridine-nucleotide reduction in locust 

 flight-muscle mitochondria was not inconsistent with the hypothesis of a 

 simple competitive reaction. 



Our doubts about the simplicity of this mechanism led us to carry 

 out an extensive study of the nature of succinate-linked pyridine-nucleo- 

 tide reduction in a variety of mitochondrial preparations under divers" 

 conditions with emphasis on the kinetics of intramitochondrial reactions. 



An example of the pyridine-nucleotide reduction by succinate in 

 rat-liver mitochondria was presented in 1956 [i] and illustrated the 

 important features of the reaction (Fig. 2). These mitochondria con- 

 taining endogenous substrate show, upon addition of succinate, increased 

 pyridine-nucleotide reduction indicated by the trace's downward deflection 

 corresponding to increased absorption at 340 m^t measured with respect 

 to 374 vcijx. The reduction rate is not rapid and a steady state is obtained 

 in about i min. The increase of respiration is not great, the initial rate of 

 0-56 /xM Oo/sec. rising to 0-9 /xM Oo/sec. Characteristic of the reduction 

 reaction is its reversal by ADP, illustrated here by the trace's abrupt 

 upward deflection upon addition of that reactant and increased pyridine- 

 nucleotide reduction upon exhaustion of the added ADP. In view of the 

 possible obscuration of DPNH reduction by the concomitant reduction of 

 TPNH in liver mitochondria [5, 12, 13] we have been studying heart and 

 kidney preparations in preference to those of liver since 1956 because of 

 their low TPNH content [13]. The percentage increase of DPN reduction 

 obtained on adding succinate to guinea-pig-kidney mitochondria runs 

 as high as fourfold, making such material ideal for kinetic and stoicheio- 

 metric studies [2]. 



Figure 3 gives an example of succinate-linked pyridine-nucleotide 

 reduction in guinea-pig-kidney mitochondria from work with Dr. 

 Hollunger [2]. Mitochondria are pretreated with 4 mM glutamate and 

 about 25",, of the total DPN is reduced in state 4. At this point addition of 

 succinate causes a striking increase in pyridine-nucleotide reduction, 

 as indicated by the large downward deflection of the trace. There appears 

 to be a transient respiratory acceleration upon succinate addition to the 

 glutamate-treated mitochondria. Thereafter net respiratory acceleration 

 on succinate addition to the glutamate-treated material is not extremely 

 large. Note that the rate at which DPN is reduced is comparable (on a 2- 

 electron basis) to the State 4 respiration rate in the presence of succinate 



