Various Forms of Yeast Lactate Dehydrogenase 



547 



cytochrome c reduced/mole flavin. The enzyme was very labile and half of 

 the activity decomposed in a few hours at 0°C. 



LDH II and III 



From many preparations only minute amounts of LDH I were obtained. 

 In these preparations LDH II constituted the main part of the LDH activity. 

 The amount of LDH III obtained corresponded roughly to the amount of 

 stable enzyme in the crude preparation. 



LDH II was completely inactivated by incubation at 55-57°C for 3 min 

 (pH 71) and by 10^ M/7CMB (0°C, pH 71). The reduction of cytochrome 

 c by LDH III was unaffected under the same conditions. The reduction of 

 ferricyanide and indophenol, however, was as labile in LDH III as in LDH 

 II. Thus, the relative activities of LDH III could be changed by heat or 

 /?CMB. The relative activities of LDH II could not be altered in this way, 

 and they remained roughly the same also during inactivation due to ageing, 

 freezing, and proteolytic digestion. This is illustrated in Table 1 . 



Table L Absolute and relattve activities of freshly prepared and of 

 partially inactivated ldh h 



The activity was determined spectrophotometricaUy at 20°C. The composition of the medium was: 

 phosphate buflfer pH 71, ionic strength 001; versene 0001 m; DL-lactate 0005 m; cytochrome 

 c2 X 10~^M, or ferricyanide to give 10~^M, or 2: 6-dichlorophenolindophenol 5 X 10~*m. 



Turnover numbers were calculated from Af at 550 m// (cytochrome c, £mM 18-6), 420 m/« (ferri- 

 cyanide, f jpM 1 6), and 600 m/( (2 : 6-dichlorophenolindophenol, £niM 10). Turnover number is defined 

 as the equivalents of acceptor reduced/mole of flavin/min. 



The sensitivity to salt was widely different for LDH II and III. The cyto- 

 chrome c-dependency curve for LDH II and its insensitivity to salts was 

 similar to that of the original crude preparation, and the cytochrome c- 

 dependency curve for LDH III and its sensitivity to salt was similar to the 

 stable enzyme of the crude preparation. In general, LDH II followed zero- 

 order kinetics down to a concentration of 10^ m cytochrome c, and the rate 

 was the same in phosphate /< 001, 008 and 0-08 + 0-04 m NaCl. LDH III, 



