562 Discussion 



in a state in which it has its own protein configuration separate from the dehydro- 

 genase moiety of bakers' yeast lactate dehydrogenase, and forms a protein complex 

 with the dehydrogenase moiety to be physiologically active. In (2) bakers' yeast 

 lactate dehydrogenase itself holds a b-type haem and a flavin. Then, if this enzyme is 

 treated at a higher pH or some other drastic conditions, the enzyme protein is modified 

 to liberate the flavin but not the b-type haem. The two models of bakers' yeast 

 lactate dehydrogenase may be considered to be as shown in Fig. 2 of our paper (Horio 

 et al., this publication, p. 557), which we have already discussed. 



Here I would like to recall again the case of succinate dehydrogenase. As is well 

 known, the extraction procedure of the enzyme is rather complex. The treatment of 

 the starting source with organic solvent such as n-butanol (Morton, Nature, Lond. 166, 

 1092, 1950) causes a serious decrease of total activity of the enzyme. However, the 

 enzyme can be easily extracted after this treatment, but not before the treatment. 

 Slater discussed with Singer at the International Symposium on Enzyme Chemistry 

 held in Japan (Proceedings of the International Symposium on Enzyme Chemistry, 

 1957) this point. The activity of succinate dehydrogenase to phenazine metho- 

 sulphate, ferricyanide, methylene blue, etc., changes during purification steps. Slater 

 questioned the inter-relationships between the activity-changes and the iron bound on 

 the enzyme. Even in a most crude state, the solubilized succinate dehydrogenase 

 cannot join with the purified cytochrome b. These things recall the same question as 

 for bakers' yeast lactate dehydrogenase: what is the native succinate dehydrogenase 

 and what is cytochrome 6? From this point of view, the activity changes of succinate 

 dehydrogenase is quite similar to the activity changes of bakers' yeast lactate dehydro- 

 genase. At the same time, there might be a great doubt about whether or not so-called 

 'cytochrome b' does exist in a free state independent from the 'physiologically native' 

 succinate dehydrogenase. This same question might be raised in relation to diaphorase 

 and cytochrome c^. In fact, during the purification of cytochrome c^ with the use of 

 cholate, an intimate connection is observed between both components, and initially 

 DPNH can reduce cytochrome c^. However, this enzymic reaction is broken as the 

 purification procedure goes on, and purified diaphorase and cytochrome c^ do not 

 react with each other. 



Very recently, Iwatsubo and Kubo (Medical School, University of Osaka, Osaka; 

 personal communication) succeeded in demonstrating an interesting flavin-level 

 reaction, using D-amino acid oxidase and xanthine oxidase which have been completely 

 purified by them. In an anaerobic condition, in the presence of both enzymes, the 

 electrons are transferred from D-amino acid to cytochrome c. This reaction may be 

 demonstrated regardless of the use of apo- or holo-D-amino acid oxidase. In fact, 

 under an experimental condition in which xanthine oxidase is perfectly stable and in 

 which flavin is not liberated from the enzyme, the apo-enzyme of D-amino acid 

 oxidase can consume oxygen in the presence of its substrate and xanthine oxidase. 

 Their experiments may illuminate the flavin-cytochrome electron transferring system 

 in respiration. 

 BoERi: With reference to the observations of Horio and co-workers, and of Nygaard, we 

 also have found that phenazine methosulphate is a good acceptor with flavocytochrome 

 b^, and it may be used in an activity test. The test may be carried out manometrically, 

 by following the oxygen consumption for reoxidation of phenazine methosulphate, 

 as used for succinate dehydrogenase by Singer and Kearney. The addition of cyanide 

 is unnecessary, as no hydrogen peroxide is formed. 



The test may also be performed spectrophotometrically by measuring the change 

 in extinction at 387 m//. We use a molar extinction of 2-27 x 10^ for the oxidized dye, 

 and of 1-62 x 10^ for the leuco compound. It is not necessary to use anaerobic 

 cuvettes but it is necessary to use air-free solutions. 



Nomenclature of Cytochrome h^ and Derived Proteins 



Morton: The various terms used in the several papers related to yeast lactate dehydro- 

 genase and cytochrome Z)^ calls for some comment, lest the confusion become too great. 



