1 68 



LARS ERNSTER 



chain phosphorylation. Then, depending on how large the electron flux is toward 

 oxygen, you may or may not get a DPN-reduction. 



Chance: I would like to congratulate Dr. Ernster on the excellent results he 

 has got in spite of the very difficult experimental problem of using acetoacetate 

 reduction as the assay for the intramitochondrial reduction of pyridine nucleotides. 

 First, we agree completely on the amytal-sensitivity, provided ATP is used as the 

 energy source. Secondly, endogenous substrate is a real factor to be considered. 

 Thirdly it wasn't clear to me in the experiments of ferricyanide reduction whether 

 or not that system had an exogenous ATP requirement. If it had I would have 

 expected it to have shown antimycin-sensitivity. 



Ernster: In the ferricyanide experiment there was no ATP added and no 

 ATP requirement. These conditions were apparently suited for producing suffi- 

 cient energy for the reduction of pyridine nucleotide, by way of the phosphoryla- 

 tion occurring between succinic dehydrogenase and ferricyanide. That phos- 

 phorylation was not sensitive to antimycin A. 



Chance: It is possible that we are in agreement on the antimycin A because 

 we could do the ferricyanide experiment in the way that requires ATP. 



TABLE I 



Effect of P, and ATP on Antimycin A-Sensitivity of 

 Reduction of Ferricyanide by Succinate 



The system contained: mitochondria from 300 mg. rat liver, 100 jumoles KCl, 

 40 |umoles glycylglycine buffer pH 7-5, 16 /j.moles MgCl.,, 20 fxmoles succinate, 

 30 /Limoles ferricyanide, i jumole KCN, and, when indicated, i ng. antimycin A, 

 in a final volume of 2 ml. Incubation for 20 min. at 30 . 



Additions (/xmoles) 



Ferricyanide reduced (/u,moles) 



ATP 



■Antimycin A 



+ Antimycin A 



15 

 60 



15 



60 



10 

 10 



10 



10-3 

 19-8 

 21 -9 

 16 -5 



22-3 

 29-7^ 



IO-6 



20 -2 



24-1 



2-8 

 22-3 



28-8^ 



* Complete reduction. 



Ernster: Yes, I can see that. Let me show a slide here which illustrates how 

 complicated this system is (Table I). It can be seen that the ferricyanide reduction 

 by succinate may or may not be antimycin A sensitive depending on the concentra- 

 tions of phosphate and ATP; and furthermore, that both phosphate and ATP 

 stimulate ferricyanide reduction, in an additive manner. I can't explain these data 

 but they do illustrate, I think, the complexity of the system. 



