KINETIC STUDIES ON THE ACTION OF 

 YEAST LACTATE DEHYDROGENASE 



By H. Hasegawa and Y. Ogura 



Department of Biophysics and Biochemistry, University of Tokyo 



Since Bernheim (1938) studied the properties of lactate dehydrogenase using 

 a crude cell free extract obtained from bakers' yeast, many investigators have 

 worked on the properties of this enzyme. Using an enzyme preparation of 

 sufficient purity, Dixon and his co-workers (Dixon and Zerfas, 1939; Bach, 

 Dixon and Zerfas, 1942, 1946) observed that cytochrome b^ was always 

 present in the enzyme system. At that time, however, they could not decide 

 whether or not the protohaem present was a prosthetic group of lactate 

 dehydrogenase itself. Five years ago, Appleby and Morton (1954, 1959) 

 succeeded in crystallizing this enzyme and established that the enzyme 

 contains two different prosthetic groups, namely protohaem and flavin mono- 

 nucleotide. The fact that the flavin mononucleotide forms an essential part 

 of the enzyme itself, was also ascertained by Boeri and his co-workers (Boeri, 

 Cutolo, Luzzati and Tosi, 1955; Boeri and Tosi, 1956). Based on their further 

 observations, they inferred that in addition to the two different prosthetic 

 groups mentioned above, eight iron ions bound to the enzyme protein seemed 

 to participate as the essential factors in the enzymic action. 



The investigation to be reported here was undertaken to obtain further 

 information concerning the mechanism of lactate dehydrogenase. Our main 

 interest was to elucidate in what manner the protohaem and flavin mono- 

 nucleotide behave as electron carriers in the mechanism of the enzymic action. 



EXPERIMENTAL 



Bakers' yeast supplied from the Oriental Yeast Co. was used as the source 

 of the enzyme. The procedures of enzyme purification were almost the same 

 as those described by Bach et al. (1946). After the cells were plasmolysed by 

 the addition of ethyl acetate, they were subjected to extraction with phosphate 

 buffer. The purification was performed by selective adsorption with calcium 

 phosphate gel followed by fractional precipitation with ammonium sulphate. 

 The enzyme samples used throughout these experiments were spectrophoto- 

 metrically free from cytochrome c. 



In the kinetic experiments, the rates of reduction of dyes were measured 

 under anaerobic conditions with a photoelectric colorimeter using Thunberg 



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