Discussion 55 



which is completely inhibited by antimycin, reacts with external sub- 

 strate only after the particles have been injured. 



Chance: One can demonstrate spectroscopically, by adding DPNH 

 to a suspension of intact mitochondria, the reduction of a pigment 

 characteristic of the microsomal fraction. Whether this is microsomal 

 contamination or whether there is naturally some of this pigment in the 

 mitochondria, is not known. But if the endoplasmic reticulum is very 

 close to the mitochondria, it would be very difficult to prove that no 

 endoplasmic reticulum contaminates the mitochondria. The "opening" 

 of the mitochondria would activate and provide a pathway from the 

 cytochrome reductase to oxygen. 



de Duve : This is a very interesting point, for if we calculate microsomal 

 contamination on the basis of glucose-6-phosphatase activity we find 

 that it is too low, by a factor of 4-5, to account for the antimycin- 

 insensitive cytochrome c reductase activity of the mitochondria. If, 

 therefore, the latter activity is due entirely to portions of the endo- 

 plasmic reticulum adhering to the mitochondria, then it follows that 

 these portions are relatively richer in reductase and poorer in glucose- 

 6-phosphatase than the bulk of the microsomes. In view of Dr. Sieke- 

 vitz's remarks, this would tend to locate the microsomal reductase in the 

 immediate neighbourhood of mitochondria within the cell. 



Slater : I would like to have thought that it was as Prof. Chance said, 

 because it is obviously simpler to have two enzymes doing the same 

 thing instead of three. However, there is other evidence that this 

 external cytochrome c reductase which is insensitive to antimycin, 

 which was discovered by Dr. Lehninger, is not due to microsomal 

 contamination, since you find exactly the same kind of behaviour with 

 heart sarcosomes (Hiilsmann, W. C, unpublished; Avi-Dor, Y., Traub, 

 A., and Mager, J. (1958). Biochim. biophys. Acta, 30, 164). There is very 

 little evidence that any microsomes containing cytochrome b^ are 

 present in the heart. 



Chance : There is more than one kind of cytochrome c reductase in the 

 cytochrome 65-containing microsomes. But one does not find cyto- 

 chrome 55-containing microsomes in the muscle even though one surely 

 has an endoplasmic reticulum. What I wanted to know was whether 

 someone has demonstrated a pyridine nucleotide cytochrome c reductase 

 in endoplasmic reticulum of muscle. Dr. Slater, you say you have. 

 Therefore, it is quite feasible that your phenomenon could have been 

 attributed to a cytochrome c reductase of the endoplasmic reticulum of 

 muscle cells which did not contain cytochrome 65. 



Slater: You now have three cytochrome reductases again, but you 

 have two of them in the microsomes ; i.e. you have two different cyto- 

 chrome c reductases in the microsomes. The evidence I mentioned 

 could only exclude the one cytochrome reductase which I know about. 

 There is also no glucose-6-phosphatase in heart homogenate. We have 

 tried it too and there is nothing corresponding to this liver microsomal 

 fraction in heart homogenate. 



de Duve : Glucose-6-phosphatase is only present in liver and in kidney. 

 Coxon: Prof, de Duve's reference to two deoxynucleases of different 



