148 Discussion 



liver mitochondria. The speed of the reaction of ADP with the inter- 

 mediates in phosphorylation is not inhibited by azide concentrations 

 of 400 ^M (Chance and Williams, 1956, loc. cit.). It is very difficult for 

 me to discuss with you now the relevance of your data on the digitonin 

 particles to our data on intact mitochondria because of this different 

 result. Does this "R factor" act on intact mitochondria? 



Lehninger: Yes, it does. Our usual assay is on digitonin particles, 

 but it produces large respiratory stimulation in intact mitochondria. 



Chance : Is it any different in effect from lack of magnesium, which I 

 have mentioned already (see p. 101)? 



Lehninger: Magnesium additions do not restore respiratory control 

 alone, nor do they affect the action of R factor. 



Estabrook: In the light of recent results (MacMurray, W. C, and 

 Lardy, H. A. (1957). J. Amer. chem. Soc, 79, 6563) on the stimulation of 

 phosphorylation by coenzyme A, I wonder whether your X might be 

 coenzyme A. Have you assayed or analysed the purified enzyme to see 

 if this nucleotide is there? 



Lehninger : When the addition of coenzyme A has had no effect, we 

 have not analysed the enzyme because we want to purify it further ; the 

 enzyme clearly depends on sulphydryl groups for activity, so we are on 

 the look-out for some connexion with Lardy' s findings. 



Estabrook : Then your results are different from Lardy's, in that you 

 do not get an effect of coenzyme A. 



Lehninger: The digitonin preparation does not respond to coenzyme 

 A, but the sonic particles that Lardy used apparently require coenzyme 

 A. 



Holton: The abstract of your paper contains the interesting state- 

 ment, Prof. Lehninger, that your R factor yields respiration rates which 

 are well beyond those which you can get with ADP or with DNP. This 

 fact cannot be explained by the reaction scheme which you have shown, 

 and necessitates the inclusion of one of the intermediate steps which 

 Prof. Slater had in his scheme. Your result with the R factor corresponds 

 with Dr. Beechey's result with magnesium on the heart sarcosome, which 

 I mentioned earlier, and seems to lead to the same conclusion concerning 

 the mechanism of formation of the DNP-sensitive intermediate in the 

 energy-transfer sequence. Would you like to tell us more about this 

 particular action of the R factor? 



Lehninger: Since our abstract was written we found that thorough 

 dialysis removed the substance which gave stimulation beyond the ADP 

 level ; its effect could be reproduced by DPN. The more highly puri- 

 fied R factor stimulates exactly to the ADP level. 



Hess : Can you get the reverse of reaction 3 from ATP, to the left-hand 

 side of your equation, with the isolated enzyme? 



Lehninger: Yes. We can start with labelled ATP and unlabelled 

 ADP e.g. in the presence of azide and digitonin fragments, where there 

 is no ATPase activity, and end up with labelled ADP (Wadkins and 

 Lehninger, 1959, loc. cit. 



Hess: We do experiments of a similar kind and find that it is very 

 advisable to preincubate the preparation with ATP so that we get, 



