On the Cytochromes of Anaerobically Cultured Yeast 233 



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DISCUSSION 



The Lactate Dehydrogenase of Yeast 



Boeri : I should like to ask Chaix for more information about the lactate dehydrogenase 

 in yeast. In our experience, different results are obtained, by varying the electron- 

 acceptor, in aerobic and anaerobic yeast. In aerobic yeast, there is an abundance of 

 a dehydrogenase which reduces cytochrome c in the presence of lactate, while the 

 strong lactate dehydrogenase activity of anaerobic yeast is not revealed by a test with 

 cytochrome c, but instead by one with ferricyanide. 



Chaix : We have found that lactate dehydrogenase activity in anaerobically grown yeasts 

 is located almost completely in the supernatant. Anaerobic pigments are not reduced 

 by DL-lactate. 



Morton: A very recent paper of Slonimski and associates reported that anaerobic yeast 

 contains a lactate dehydrogenase which is a flavoprotein (free from haem) specific for 

 d(— ) lactate, whereas the enzyme in aerobic yeast, the haemoflavoprotein, is specific 

 for l(+) lactate. Could Chaix indicate as to where is the enzyme in the aerobic yeast? 



Chaix: We have used DL-lactate with aerobically and anaerobically grown yeast. Con- 

 cerning your second question, we have found lactate dehydrogenase activity only in 

 the mitochondrial fraction in aerobically grown yeasts. In this case the cytochromes 

 are reduced by DL-lactate. 



Components of the Respiratory Chain in Yeast Mitochondria 



Chance: It is important to emphasize the need for distinguishing between pigments of 

 the respiratory chain and accessory pigments. An unusually effective way of doing 

 this is to observe the cytochromes involved in the steady state of aerobic metabolism, 

 since the existence of the steady-state reduction of a cytochrome in intact cells and in 

 phosphorylating mitochondria is acceptable as preliminary evidence for its function 

 in electron transfer. Accurate observation of steady states requires the difference 

 spectrum technique: the oxidized minus the steady-state oxidized spectrum. It has 

 recently been possible in studies with W. Bonner to obtain this steady-state difference 

 spectrum at liquid nitrogen temperatures (see also Estabrook, this volume, p. 436). 

 Thus the method simultaneously gives a clear identification of the kind of cytochrome 

 and its function in electron transfer. 



Two examples are given, one indicating respiratory enzymes of baker's yeast cells 

 (Fig. 1 (p. 234)), the other of ox heart mitochondria (see Fig. 3, Chance, this volume, 

 p. 607). Both these records show clearly what other methods fail to do: that cyto- 

 chrome Cj is reduced in the steady state and hence functional in electron transfer in the 

 intact cell and isolated mitochondria. 



Chaix : The experiments mentioned by Chance, which aim to distinguish between pigments 

 which belong to the respiratory chain and the other pigments, are very interesting. 



But in order to correlate his observations with ours we would first have to agree 

 on the definition of the term 'respiratory chain'. It should be possible, for instance, 

 to postulate that cytochrome c functions as an electron-carrier in both aerobic and 

 anaerobic electron-transfer. 

 Chance considers this question from quite a different point of view from ours. 



On the ^Haemoglobin' Absorption Bands of Yeast 



Lemberg: Are the bands at 575 m/t and 630 m/t observed by you in yeast abolished by 

 dithionite? If they are resistant to dithionite, the former may be due to a cryptohaem 

 a-cytochrome, the latter to a cytochrome of type a^. 



