Discussion 49 



the nucleus, the mitochondria and the cytoplasmic elements in order to 

 determine whether this would abolish or enhance metabolic regulation. 

 We observed that the interior of the cell had been disrupted, i.e. the 

 nucleus, mitochondria and fat bodies had moved to such an extent that 

 we considered tliis as rather a dramatic effect. Then we took a portion 

 of the cells to see whether the metabolic regulation caused by glucose 

 addition would still function : it did. Thus, if there is c*, cytoskeleton 

 which is relevant to the interactions of glycolysis and oxidation, it is 

 one which has a considerable degree of mechanical stability. 



Siekevitz: I can answer this by alluding to the old controversy of 

 whether there is RNA in the mitochondria. If you isolate mitochondria, 

 take a picture and develop it, you find a good many of these mito- 

 chondria still encapsulated within some endoplasmic reticulum mem- 

 branes, a portion of which has come down with the mitochondria. It is 

 very difficult to wash out these membranes; in fact, it just cannot be 

 done. We are left with the controversy and with the problem of the 

 degree of stability, contactness or "togetherness", which exists between 

 mitochondria and microsomes. 



Chance: I think there is a lot to be said in your favour, from the 

 standpoint of the permeability of isolated mitochondria to added pyri- 

 dine nucleotide. They are apparently impermeable. Is that due to the 

 fact that they are prepared in "sealed packages" enveloped in the 

 endoplasmic reticulum? I do not believe that anybody has proved this 

 but it should be considered here. 



King: Dr. Siekevitz, in your Table I the ratios 



specific activity of mitochondria 

 specific activity of mitochondrial membrane 



are about the same for succinoxidase and cytochrome oxidase, but quite 

 different for DPNH cytochrome c reductase. I wonder why. Secondly, 

 how have you tested succinoxidase, cytochrome oxidase and DPNH 

 cytochrome c reductase? 



Siekevitz : We tested by the usual procedure ; succinoxidase with added 

 cytochrome c and also in the presence of calcium and aluminum salts ; 

 cytochrome oxidase by the oxidation of reduced cytochrome c; and 

 DPNH cytochrome c reductase by DPNH oxidation, i.e. by the usual 

 spectrophotometric or manometric means. We break up the mito- 

 chondria with detergent, which is a fairly easy process. All we can say 

 is that perhaps the DPNH cytochrome c reductase was once on the 

 membrane, but it is much more easily broken off than is the succinoxi- 

 dase or the cytochrome oxidase. We have no other explanation of why 

 that enzyme was in the supernatant from the broken up mitochondria, 

 while the other enzymes could be sedimented with the membranes. 



