54 Discussion 



have carried out experiments with the enzyme, rhodanese, similar to 

 those of Prof, de Duve and his colleagues. After consultation with Prof, 

 de Duve and Dr. Bendall we carried out an experiment very similar to 

 one which Prof, de Duve has described, the effect of hypotonicity — 15 

 minutes at 0° with various concentrations of sucrose — on the activity 

 of mitochondrial rhodanese. The curve we got is almost identical with 

 one obtained by Prof, de Duve. Obviously, then, the rhodanese in 

 mitochondria behaves in that respect similarly to the glutamic and 

 malic dehydrogenases. An interesting point is that all measures which 

 make the mitochondria swell seem to make the rhodanese active, e.g. 

 addition of thyroxine, phosphate, oxidizable substrates, etc. We have 

 tried the action of various combinations of these agents on swelling and 

 we can match the swelling effects with the effects on rhodanese. 



de Duve: I am very interested in this work of Drs. Greville and 

 Chappell. It is particularly interesting because the substrates of 

 rhodanese are so small. Here, we are dealing with very small ions, and 

 if the swelling effect causes activation it could perhaps be due to the 

 fact that these ions can penetrate swollen mitochondria but not shrunken 

 ones. 



In our laboratory. Dr. Bendall has tried to verify whether there is a 

 state of osmotic swelling of the mitochondria where the membrane has 

 become permeable to the substrates of glutamic dehydrogenase, without 

 however allowing the enzyme to leach out, and from which reversal 

 to the original lack of permeability is still possible in isotonic medium. 

 He has obtained some indications that this might occur but his results 

 were not conclusive. 



Siekevitz : For purposes of metabolic control, do you think from your 

 experiments that since each of the enzymes comes out at a slightly 

 different peak, that might be an indication that one enzyme is in one 

 particle and another one in another particle? Is there any way of 

 separating them? 



de Duve: We can separate crude subfractions in which the ratios of 

 two enzymic activities differ to some extent, but the meaning of this is 

 not clear. To verify your hypothesis, one would have either to effect a 

 fairly clearcut separation of the two activities or to measure the enzymic 

 content of single particles. We are a long way from there. 



Estabrook : Have you studied the distribution of DPNH cytochrome c 

 reductase in the same manner as you did cytochrome oxidase? The 

 question comes from Dr. Siekevitz's paper as to whether the 65 enzyme 

 of the DPNH cytochrome c reductase which is antimycin-insensitive is 

 always associated with mitochondria. Your distribution studies would 

 perhaps answer that. 



de Duve : According to the investigations performed in our laboratory 

 by Dr. Pressman, liver microsomes contain a cytochrome c reductase 

 which is entirely insensitive to antimycin and accounts for approxi- 

 mately 70 per cent of the total activity. The remainder is associated 

 with the mitochondria in what appear to be two distinct forms. About 

 one-half of the mitochondrial activity appears to be readily accessible 

 to external substrate and is insensitive to antimycin; the other half. 



