560 Discussion 



Because our crystalline lithium salts of these two acids occur as monohydrates, and 

 infra-red spectroscopy by Bellamy and Williams (Biocliem. J. 68, 81, 1958) failed to 

 detect the presence in either solid salt of a keto group or of a molecule of water of cry- 

 stallization, we tentatively suggested a hydration of the keto- to a diol-form as part of 

 the enzymic reaction. While this is still not impossible, recent studies kindly carried out 

 by D. A. Long (of University College, Swansea) on the Raman spectra in aqueous 

 solution do not support the above findings based on the solid substances; in fact they 

 indicate predominantly a keto-form in aqueous solutions of lithium or sodium pyruvate. 

 So far, fluorescence difficulties have prevented similar observations on hydroxypyruvate 

 solutions. 



Properties of Intact and Modified Cytochrome b^ 



Nature of Bakers' Yeast Lactate Dehydrogenase 



By T. HoRio (Osaka) 

 HoRio: I would like to consider again the true physiological function of bakers' yeast 

 lactate dehydrogenase. Of course, the enzyme extracted and purified from bakers' 

 yeast can dehydrogenate lactate and other substrates: namely, a-hydroxybutyric 

 acid, and exceptionally TPNH. When these substrates are added to the enzyme 

 solution, the cytochrome Z)2-moiety bound on the enzyme is reduced. Of the various 

 substrates tested, lactate is most rapidly attacked by this enzyme, so most workers use 

 lactate as its substrate. However, using cytochrome c in its reduced form as an electron 

 donor, the back reaction from pyruvate to lactate has never been demonstrated to take 

 place physiologically, as reported by Boeri and co-workers. Furthermore, any meta- 

 bolic pathway to produce lactate has never been found in bakers' yeast, as far as I 

 know. From this point of view, I am just wondering the reason why bakers' yeast 

 cells have such a lot of lactate dehydrogenase. 



When purified, bakers' yeast lactate dehydrogenase is quite water-soluble. However, 

 our procedure for its extraction from bakers' yeast cells is very complex and different 

 from that of other workers. In our experiments, pressed bakers' yeast is disrupted 

 by a minimum amount of ethyl acetate, then washed with a great deal of water. The 

 washed cells are extracted with 20 %-saturated ammonium sulphate at a neutral pH. 

 Most of cytochrome c can be extracted in the salt solution by this extraction, but 

 almost none of the enzyme by the procedures up to this step. This debris is suspended 

 again in water, then the enzyme comes out of the debris to a great extent. 



When the activity of bakers' yeast lactate dehydrogenase purified mainly according 

 to the method of Bach, Dixon and Zerfas (Biochem. J. 40, 229, 1946) is assayed by 

 the phenazine methosulphate-method of Singer and co-workers, and by methylene 

 blue-, ferricyanide- and cytochrome c-methods with the use of lactate as the substrate, 

 the ratio of the phenazine methosulphate activity to the methylene blue, etc., activities 

 varies for diff"erent enzyme preparations. As a rule, we might say that the more native 

 the enzyme is, the lower is the ratio of phenazine methosulphate activity to methylene 

 blue activity (see Fig. 1, Horio, Yamashita, Yamanaka, Nozaki and Okunuki: this 

 volume, p. 555). 



As we have already pointed out, the modification of yeast lactate dehydrogenase 

 which occurs on various treatments suggests to us the scheme of electron transport 

 which may be broken at different sites (see Horio et al., this volume, p. 554). The 

 intimate junction between the 'dehydrogenase' and 'cytochrome b.," moieties may be 

 broken at the lower and high pH ranges, and the reaction (4) may take the place of 

 reaction (2). 



It may be thought that the differences of the results presented by Ogura, Nygaard, 

 Boeri, Morton and ourselves are dependent upon these changeable properties of the 

 enzyme itself, as well as the original source of bakers' yeast cells as suggested by 

 Nygaard. To my regret, I have not yet succeeded in crystallizing this enzyme although 

 I have used four kinds of yeast cells made in Japan, and although our extraction 

 method works well with all these yeasts. Therefore, we have been compelled to use a 

 rather crude enzyme preparation for this experiment. 



