THE FUNCTIONAL LINK OF SUCCINIC DEHYDROGENASE 205 



give a short period to allow the "succinate" to enter the cell before adding the 

 inhibitor, the succinate that disappeared from the medium before the inhibitor 

 was added subsequently becomes oxidized in the ceil at almost the normal rate. 

 If, however, you break the membrane before adding these inhibitors of "suc- 

 cinate" transport, succinate oxidation is not inhibited. There is not time to go 

 into details, but we have good reasons for believing that succinate goes through 

 the membrane either as succinyl-CoA or as succinyl-lipoate or some closely 

 related substance. Thus, the oxidation of succinate requires its prior "activation" 

 to allow it to reach the oxidation systems. We believe that the "activation" step 

 is catalyzed by an enzyme located in the plasma membrane. 



Hess : I would like to ask a short question to one of the last three speakers 

 about the arsenate treatment; according to Dr. Ernster the pyridine nucleotides 

 were largely reduced after the arsenate treatment. How do you explain it ? And 

 what is the reductant ? 



Packer: I would like to comment on some recent experiments which Dr. E. E. 

 Jacobs and I have been doing, as they may have some bearing on this problem. 

 We have been looking at certain shunts of electron transport involving the oxidase 

 end of the chain. For example, we use ascorbate as reducing agent and catalysts 

 such as tetramethylphenylenediamine ; we have been able to show that these shunts 

 can be tightly coupled to phosphorylation and show respiratory control with ADP 

 and thus can calculate P to O ratios by measuring O., utilization (polarographically). 

 If the mitochondria are carefully washed the endogenous substrate can be removed 

 so that P:0 ratios come out to be about 10. It happens that this shunt is capable 

 of reducing pyridine nucleotides, and in the steady state the pyridine nucleotides 

 can be oxidized and reduced by initiating a brief cycle of phosphorylation by 

 adding a small amount of ADP. The oxidation through the shunt is not inhibited 

 by antimycin but the reduction of pyridine nucleotide is completely blocked in 

 the presence of this substance. I would commend this shunt as of possible interest 

 in connection with the phenomenon of reversal of electron transport just discussed. 



Estabrook: I should just like to state that in particles which we know are 

 devoid of endogenous substrate, DNP preincubation does not inhibit succinate 

 oxidation. This is a situation with particles which will give good phosphorylation 

 in the absence of DNP and also a situation where pyridine nucleotide is reduced 

 by succinate. This is one more additional piece of evidence for the complexity of 

 the endogenous substrate. 



Azzone: I don't see why this should be. 



Estabrook: You didn't produce any oxaloacetate. 



Chappell : The same is true in kidney mitochondria which has very little 

 endogenous substrate. 



Azzone: We think that the ATP requirement for succinate oxidation is present 

 only above a certain level of structural integrity. If we take away, by destroying or 

 fragmenting the mitochondria, the structural barrier which makes necessary the 

 energy-requiring reaction then we find neither an inhibition nor an ATP stimula- 

 tion of succinate oxidation. 



Chappell: This is not true in kidney mitochondria which responds to normal 

 respiratory control and P:0 ratios, and they are just free of endogenous substrate. 

 The addition of oxaloacetate or malate will induce all the phenomena which you 



