Discussion 13 



mainly outside the mitochondria and, secondly, there is the compli- 

 cation that any citrate which accumulates upsets the balance of free 

 magnesium ions. As you know, it has been suggested by Raaflaub 

 (1956, Helv. physiol. pharmacol. Acta, 14, 304) that among the rate- 

 controlling factors are magnesium ions, which are needed for a variety 

 of enzymes, and that phosphate, ADP and ATP all react through their 

 magnesium binding. The complexing capacity of ATP is very much 

 greater — by a factor of 50 — than the complexing capacity of ADP. 

 Citrate has also a complexing capacity. This has been put forward as a 

 possibility. I have done a few experiments on this, and they do not 

 support it very well. But it has to be borne in mind that it is not easy 

 to make sure that you get your chelating agent into the mitochondrion. 



Dickens: In the case of the mitochondrial citrate oxidation, a very 

 interesting survey has been made recently by Ernster and Navazio 

 where it was found that the DPN-specific isocitric dehydrogenase in rat 

 liver is localized exclusively in the mitochondria, although there is 

 some TPN-linked dehydrogenase there too (Ernster, L., and Navazio, 

 F. (1957). Biochim. biophys. Acta, 26, 408). The TPN one is, in fact, 

 more active in mitochondrial reduction of coenzyme. But owing to the 

 extremely inefficient transport systems for reoxidizing TPNH the DPN 

 one wins in the overall oxidation of isocitrate. According to their figures, 

 which looked rather convincing, the greater part, about 75 per cent, of 

 the oxidation of tsocitrate in mitochondria of liver should go through 

 DPN and only 11 per cent via transhydrogenase and 7 per cent via TPN. 

 This case is an interesting example where you not only have the effect of 

 substrate concentration, but the choice of which enzyme-coenzyme 

 pathway the oxidation will take. 



de Duve: To return to Prof. Potter's question, I think Sir Hans was 

 referring to control mechanisms occurring in systems where the enzyme 

 concentration is constant during the experiment. Prof. Potter was 

 referring to a fairly slow process of adaptation. We have to keep in mind 

 another possibility e.g. where an enzyme is very rapidly converted into 

 an inactive form and can be very rapidly reconverted into an active 

 form, as is known to be the case for phosphorylase. This may occur 

 with other enzymes. 



Krebs : This would be the type of case where the hormonal influence 

 is of importance. The activity of phosphorylase depends on the presence 

 of adrenaline. 



de Duve : Yes, but several other factors besides adrenaline are known 

 to influence the phosphorylase system. The work on muscle, for 

 instance, indicates that the activity of phosphorylase is also under 

 intracellular control and varies with the state of the tissue. 



Slater : With reference to Prof. Dickens' remark on the mechanism of 

 oxidation of isocitrate. Dr. Purvis in our laboratory has recently 

 investigated this question with both liver and heart mitochondria 

 (Purvis, J. L. (1958). Biochim. biophys. Acta, 30, 440). His conclusion 

 does not agree with that of Dr. Ernster, but supports the view of Kaplan, 

 namely that the oxidation of tsocitrate does go through the TPN and 

 the pyridine nucleotide transhydrogenase. If the mitochondria are 



