14 Discussion 



really depleted of pjrridine nucleotide — and this must be controlled by 

 direct analysis — then isocitrate is only oxidized on the addition of both 

 DPN and TPN. 



Dickens : That is most interesting. According to Ernster's figures the 

 pyridine nucleotide transhydrogenase could undertake a maximum of 

 about 1 1 per cent of the total oxidation. I do not understand how the 

 soluble tsocitric dehydrogenase which, I believe, accounts for 90 per 

 cent of the total activity comes into the story. Is it that the isocitric 

 oxidation can take place in the soluble fraction and then the products 

 pass back into the mitochondria? 



Siekevitz: It has been observed (Reinafarje, B., and Potter, V. R. 

 (1957). Cancer Res., 17, 1112) that the TPNH-cytochrome c reductase 

 is solubilized very easily from mitochondria, and it is possible that the 

 soluble isocitric dehydrogenase is really a mitochondrial enzyme ; it may 

 also be very easily solubilized, i.e. during the preparation of the mito- 

 chondria there is a loss of this enzyme. 



Dickens : In fact you suggest that it is an artifact. 



Pardee : As regards the quantities of enzymes in systems of the type 

 you are discussing, there still remains a problem of metabolic control. 

 Presumably these enzymes are in surplus over the amounts required for 

 the reactions to be carried out at physiological rates. One may ask 

 what limits the amounts of enzymes in animal systems. How much 

 enzyme is made, and why is a certain amount made? If there were too 

 few enzyme molecules for any step they would be rate-limiting. The 

 fact that the amounts of many enzymes do not seem to be rate-limiting 

 in animal tissues presumably means that there is something that makes 

 them not rate-limiting, i.e. in excess. 



Krebs : Many enzymes are present in quantities in excess of ordinary 

 requirements. There must be some enzymes that are, in fact, rate- 

 limiting. 



Pardee : My question is not how greatly in excess they are, but is there 

 a mechanism that provides for the enzymes in only a small excess, or 

 which does limit the amount; i.e. a mechanism which limits the forma- 

 tion of these enzymes? In animal tissues such mechanisms may not be 

 easy to observe because of the animal's homeostatic system. 



Dickens: That is certainly a very complicated situation. Perhaps in 

 major pathways there may be no circumstances in which the amounts 

 of enzyme are limiting ; but when it comes to minor ones, e.g. the pentose 

 phosphate pathway, they may very well be limiting factors, and the 

 amount of enzyme is probably quite a considerable factor in some 

 circumstances in deciding, e.g., the extent to which the pentose or 

 hexose monophosphate oxidative pathway is, in fact, occurring. This is 

 a typical branching of pathways, in which one regulatory factor must be 

 the relative amounts of the enzymes, as well as the coenzymes, of the 

 hexose monophosphate and glycolytic systems. 



Krebs : It is in stages where pathways are initiated that the enzyme 

 amount must be the important part. In general, intermediate products 

 never accumulate. That must mean that there is enough enzyme to 

 cope with the intermediates as they are formed. We have long been 



