88 Discussion 



Slater : At the present stage of our work, we should not hke to make a 

 final decision as to whether our compound is a DPN~^I or DPNH'--'I 

 compound. There are many observations which we and others have 

 made which are easier to explain on the basis of a DPN ^--'I compound 

 than of a DPNH'^I compound. But I should not say that they were 

 conclusive. At the moment we should say that we have evidence for an 

 intermediate precursor of DPN or DPNH. I would not like to go so far 

 as to say that this is certainly a DPN and not a DPNH compound. We 

 hope to settle the question by isolating the compound. 



As Dr. Racker has pointed out, it is certainly possible to have a 

 DPN~I compound which absorbs maximally at 340 m^x. 



Siekevitz: Some evidence has been published that the initial phos- 

 phorylation takes place between DPNH and flavoprotein by means of a 

 direct phosphorylation of the flavoprotein, and that a loose complex 

 might be formed between DPNH and flavoprotein if inorganic phosphate 

 is around (Low, H., Siekevitz, P., Ernster, L., and Lindberg, O. (1958). 

 Biochim. biophys. Acta, 29, 392). I wonder if your inhibited compound 

 might either be a DPNH phosphate — a cyclic phosphate — or a DPNH 

 flavoprotein. 



Slater: Dr. Ernster's theory, as I understand it, is that a reduced 

 flavoprotein phosphate is the intermediate ; and that is rather different 

 from what we are suggesting here. He suggests a reaction of DPNH 

 plus the flavoprotein plus inorganic phosphate which gives reduced 

 flavoprotein phosphate plus oxidized DPN ; so he has no intermediate of 

 the DPN, which is what we are studying. 



Siekevitz : He also thinks that initially there is a charged ion complex 

 formation between DPNH and flavoprotein. 



Slater : On the evidence of these experiments alone, anything like that 

 is possible. We do not think it is so, on the basis of other experiments 

 which lead us to believe that phosphate comes into the series of reactions 

 rather later. 



Lehninger: In the course of our own work on the mechanism — as I 

 will explain later — we have also been searching for "high-energy" 

 derivatives of DPN. One point which came to our attention very 

 forcibly is that not all the DPN molecules in the mitochondrion may be 

 of the same chemical species (Devlin, T. M. (1959). J. biol. Chem., in 

 press; Gamble, J. L., Jr., and Lehninger, A. L. (1956). J. biol. Chem. 223, 

 921). Prof. Chance has also presented evidence that there is some 

 functional compartmentation. Our evidence derives from studying 

 the digitonin fragments of the mitochondrial membrane. There, the 

 bound DPN seem to have quite different characteristics from the total 

 DPN of intact mitochondria. This brings up the suggestion that pos- 

 sibly only a few DPN molecules out of the large number in mitochondria 

 may be directly connected with each respiratory chain ; the others might 

 feed into it via a DPNH — DPN transhydrogenase. The bound DPN of 

 the submitochondrial fragments is not discharged by phosphate. 



Prof. Slater, have you followed the extramitochondrial DPN during 

 some of your experiments? Hunter found discharge of DPN from mito- 

 chondria with phosphate, and recently he reported that he can rebind 



