Actio/! of Yeast Lactate Dehydrogenase 535 



tubes, whereas those of cytochrome c and ferricyanide were determined 

 spectrophotometrically. Prior to the experiments, the concentrations of 

 dyes were determined by careful titration with a standardized dithionite 

 solution under anaerobic condition. 



In most of the experiments racemic lactate was used as the substrate. The 

 concentrations of substrate described in the data were those of L-lactate 

 contained in the reaction mixtures, since it has been confirmed that D-lactate 

 did not show any effect on the enzymic reaction, as mentioned by Boeri et al. 

 (1955). 



RESULTS AND DISCUSSION 



Properties of the Enzyme 



The spectrum of the enzyme, reduced by the addition of lactate, showed 

 maxima at 557, 528 and 423 m//; the Soret maximum of the oxidized form 

 was at 412 m/<. These results were in good agreement with those reported 

 by other authors (Appleby and Morton, 1954, 1959; Boeri et al, 1955). 



The spectrum of flavin mononucleotide attached to the enzyme protein 

 was not observed by direct spectrophotometry, since it was largely covered 

 by that of cytochrome b^- Using a tonometer-type chamber reported by 

 Nakamura (1958), we could measure the spectrum of the former compound 

 by the following procedures. After the air in the chamber was evacuated and 

 replaced by nitrogen gas, a small amount of lactate (final concentration about 

 50 /m) was added to the buffered enzyme solution placed in the main cham- 

 ber. The complete reduction of cytochrome bo was checked spectrophoto- 

 metrically, and then the enzyme solution was titrated carefully with an 

 oxygen-free methylene blue solution. When the blue colour of the oxidized 

 form of the dye appeared shghtly, the titration was stopped and the mixture 

 was allowed to stand for several minutes. Figure 1 shows the difference 

 spectrum between the enzyme solution thus treated and the reduced one (in 

 which both the protohaem and flavin mononucleotide had been completely 

 reduced). The spectrum, which showed two maxima at 385 and 445 m/v, 

 seemed to be due to the oxidized form of flavin mononucleotide. Conceivably, 

 the flavin mononucleotide moiety of the enzyme was oxidized by the treat- 

 ment described above, while the protohaem moiety remained in a reduced 

 state. Assuming that the millimolar extinction coefficient of flavin mono- 

 nucleotide attached to the enzyme protein was the same as that of the free 

 molecule, its concentration in the mixture was estimated to be 16-4 /iM from 

 the value of eox — Cred = 9-0 cm~i mM~i. Since the concentration of cyto- 

 chrome Z)2 was found to be 18-6 /^m, it was deduced that the molecular ratio 

 of flavin mononucleotide to protohaem present in one molecule of enzyme is 

 1 : 1 as reported by Appleby and Morton (1954). 



The effect of riboflavin upon the enzymic reaction was investigated as 



