146 Discussion 



Racker: Could this phenomenon be due to a very deUcate balance 

 between phosphorylation and ATPase activity or is this possibility out 

 of the question? 



Lehninger : We cannot exclude an action of an ATPase but it seems 

 extremely unlikely. The effect cannot be simulated by increasing 

 ATPase activity. Furthermore, it would be necessary then to postulate 

 that the presence of ADP can inhibit ATPase almost completely. 



Racker: Pullman, M. E., and Penefsky, Z. J. in our laboratory 

 (1958, Arch. Biochem., 76, 227), have succeeded in separating two 

 distinct fractions from beef heart mitochondria after breakage with 

 glass beads in a Nossal shaker. These fractions appear to be different 

 from those described by Prof. Lehninger. There is one fraction which is 

 particulate and catalyses respiration but does not couple it to phos- 

 phorylation. The second factor is soluble and has been purified by 

 ammonium sulphate and protamine fractionation. This factor when 

 added to the particulate fraction allows phosphorylation to be coupled 

 to oxidation. Oxidative phosphorylation in this system is sensitive to 

 dinitrophenol. Curiously enough, the soluble fraction also contains a 

 dinitrophenol-sensitive ATPase activity. Whether this activity is in 

 any way associated with the phosphorylation activity we cannot tell 

 at the present time, but at first sight this soluble factor appears to be 

 different from Prof. Lehninger's fractions, and I am at a loss to fit his 

 and our findings into a common scheme. 



Lehninger: It is possible that this fraction represents the whole 

 coupling machinery, including X. 



Racker : You mention that the complete system is very unstable and 

 difficult to purify. The soluble fraction is quite stable, particularly in 

 ammonium sulphate. 



Lehninger: I would have expected that the second step where ^^p 

 enters would be unstable. 



Racker : The soluble factor, after several steps of purification, does not 

 catalyse the ^^Pj exchange and preliminary experiments indicate that 

 it has little if any ADP exchange. 



Lehninger : That is very puzzling. 



Siekevitz: With regard to the R factor, is there any bound ADP 

 there ; could any bound ADP have been released from the particles? 



Lehninger: We have not examined that closely. However, the 

 preparations are exhaustively dialysed. 



Siekevitz : Where do you visualize the magnesium activation to occur 

 in ATPase? Would it be in the last step or the one before? 



Lehninger: The action of magnesium seems to be clearly different. 

 However, magnesium-stimulated ATPase is also inhibited by azide. 

 We feel that reaction 2 is common to both kinds of ATPase ; but there 

 must be something different between the action of magnesium and 

 DNP. 



Slater : One of the many points I would like to discuss with you is that 

 of the azide inhibition. Following the work of Robertson and Boyer we 

 studied also the effect of azide on the ATPase (Robertson, H. E., and 

 Boyer, P. D. (1955). J. biol. Chem., 214, 295). We found two effects of 



