Bakers' Yeast Lactate Dehydrogenase 553 



form at 413 m/t (y-band). This enzyme was called cytochrome b,^, by Bach, 

 Dixon and Zerfas (1946) who partially purified the cytochrome simultane- 

 ously with increasing the specific enzymic activity. On the other hand, the 

 cytochrome /?2 crystallized by us (Yamashita et al., 1957) contained one haem 

 iron atom in each 22,000 g protein, but no flavin nor non-haem iron. More- 

 over, it showed no enzymic activities in all tests tried in the presence and 

 absence of flavin (flavinadenine dinucleotide, FAD ; riboflavin phosphate, 

 FMN; and riboflavin, RF). Hereafter in this paper, in order to avoid 

 confusion, the enzymic and non-enzymic cytochromes will be called Y-LDH 

 and cytochrome Z^g, respectively. 



In addition to the method of Yamashita et al. (1957), by which cytochrome 

 b^ was directly extracted at pH 8-0 from the cells of bakers' yeast previously 

 disrupted by ethyl acetate, it was found that cytochrome b^ could be split 

 from the Y-LDH purified according to the method of Bach et al. (1946) by 

 treating it at high pH. 



The present paper deals with some studies on relationships between the 

 lactate dehydrogenase moiety and the cytochrome Z^g rnoiety, both of which 

 seemed to be the main moieties of the whole enzyme, Y-LDH. 



ENZYMIC ACTIVITIES OF YEAST LACTATE 

 DEHYDROGENASE (Y-LDH) 

 The preparation of Y-LDH purified mainly according to the method of 

 Bach et al. (1946) has been found capable of oxidizing various substances 

 other than lactate: a-glycerophosphate (Lehmann, 1938), glycerate (Dickens 

 and Williamson, 1956), malate and reduced triphosphopyridine nucleotide, 

 TPNH (Yamanaka, Horio and Okunuki, 1958), and a-hydroxybutyrate 

 (Yamashita, unpublished). If these substrates are added to the enzyme 

 preparation purified to a specific activity of about 6,000 units (as Q^q at 

 30°C) Y-LDH immediately exhibits its reduced absorption spectrum to a 

 variable extent, dependent on the substrate. With equal concentrations of 

 the substrates, the extent of reduction is highest with lactate, somewhat 

 lower with a-hydroxybutyrate, and very low with the others. The reduction 

 by malate gradually increases as its concentration rises to the maximum 

 examined (0-5 m). On the other hand, Y-LDH is known to be able to deliver 

 electrons from the substrates to cytochrome c, methylene blue, ferricyanide, 

 phenazine methosulphate, etc. The rates of reduction of these electron 

 acceptors by the substrates vary in proportion to the extent to which Y-LDH 

 itself is reduced by these substrates. 



Relationship between Y-LDH and Cytochrome b^ 



During the purification of Y-LDH, the sample is always contaminated 

 with the cytochrome b^ not bound to Y-LDH. If lactate is aerobically added 

 in excess to such an enzyme preparation, all Y-LDH is immediately reduced. 



