68 F. EDMUND HUNTER, JR. 



Discussion 



Williams : May I make one general comment ? I am interested in the kinetics 

 of the swelling that you get and I have a feeling that we in the mitochondrial field 

 should be thinking about this type of kinetics which is more familiar in the erythro- 

 cyte field where you regularly find S-shaped progress curves which represent the 

 integral form of the Gaussian distribution of red cell fragilities. If we had better 

 methods than just optical density measurement for following lysis then we might 

 observe this more often. We have observed such curves in measuring the onset of 

 choline oxidation in mitochondria which is related to the integrity of the mito- 

 chondrial membrane. It might even be useful to use them to measure the homo- 

 geneity of a population of mitochondria. 



DiscHE : What was the technique of homogenization which you used in studying 

 the distribution of different particles after ascorbate treatment, and in what 

 medium were the mitochondria suspended when you treated them with ascorbate ? 



Hunter: We used 0-33 m sucrose plus 0-025 m tris buffer pH 74 in all these 

 experiments. In the protein distribution experiments the mitochondrial lysis 

 experiment was carried out in this medium in the usual fashion and then the 

 suspension was subjected to differential centrifugation and separated into four 

 fractions, 8000 x g, 20 000 x g and 100 000 x g pellets plus the supernatant. 



Dische: The distribution of fractions in the non-treated mitochondria was 

 simply determined by centrifugation of your suspension of mitochondria ? 



Hunter: We have centrifuged the suspensions at o and at 25" under exactly 

 the same conditions but in the absence of ascorbate. 



Dische: Would you not suspect that under these conditions your population 

 of mitochondria was very inhomogeneous, because you have already got a certain 

 distribution on your original suspension ? 



Hunter: Not entirely, although it undergoes some change. Actually we think 

 that the largest change is due to dilution. Mitochondria when diluted out as they 

 are for experiments like this do begin to undergo changes. We have electron 

 microscope evidence for this. If you take a mitochondrial suspension and recentri- 

 fuge it without much dilution you will get far more than So",, of the protein in the 

 so-called mitochondrial pellet. If you dilute it out even at o you get some change 

 just by that dilution, and you get only 80% of the protein in the mitochondrial 

 fraction and an appreciable portion in the soluble fraction, which I am sure is not 

 all a contaminating soluble protein. 



Weinbach : Was the ascorbate effect you noted preceded by the loss of pyridine 

 nucleotide, in other words did you examine what happened during the lag phase ? 



Hunter: Within the limits of methods we use we could not say that it preceded 

 this, but it occurs simultaneously with it. 



Hess : Do you know what the fate of ascorbate is and how the kinetics are ? 

 How does it compare to the swelling action ? 



Hunter: I can't answer that question exactly. We were interested in what 

 happened to the ascorbate during the lag period. We were somewhat amazed when 

 the preliminary experiments indicated that it was not disappearing, yet later 

 experiments with an oxygen electrode indicated an oxygen consumption. That 



