554 T. HoRio, J. Yamashita, T. Yamanaka, M. Nozaki and K. Okunuki 



but the free cytochrome b.^ is only shghtly reduced because of its high aut- 

 oxidizabiUty (Yamashita, Horio and Okunuki, 1958). Most of the free 

 cytochrome b^ can be removed from tiie enzyme preparation by taking 

 advantage of different affinities of calcium phosphate gel for the two proteins. 



Reductions of Cytochrome c and Cytochrome bo by Lactate, oi-Hydroxybutyrate 

 and Malate in the Presence of Y-LDH 

 Under anaerobic conditions at pH 6-4, cytochrome c is reduced by Y-LDH 

 42 times as fast as cytochrome b^. When cytochrome c or cytochrome b^ is 

 used as a final electron acceptor of Y-LDH, reduction of cytochrome c by 

 lactate, a-hydroxybutyrate, and malate shows optimal pH at pH 6-5, 5-5 

 and 8-0, respectively, while reduction of cytochrome b^ shows optimal pH at 

 about 5, 5, and 8-9, respectively. With lactate and a-hydroxybutyrate, 

 activity-pH curves for the cytochrome c reduction reactions are fairly sharp 

 and roughly symmetrical on both sides of each optimal pH. However, the 

 activity-pH curves for the reduction of cytochrome b^ by lactate and oc- 

 hydroxybutyrate are asymmetrical, showing appreciable activity even at 

 pH 4-0 on the acid side of the pH optimum, but dropping steeply on the 

 alkaline side. In contrast, the activity-pH curve for the reduction of cyto- 

 chrome b^ by malate is nearly constant between pH 8-9, and falls steeply at 

 pH lower than 8. The remarkable differences between the activity-pH 

 curves for the reduction of cytochrome c and cytochrome b^ by the various 

 substrates might be explained by the following pathway: 



(Y-LDH) Most easily damaged here 



T 



Dehydrogenase moiety y^ ^^^ > Cytochrome b^, moiety > Cytochrome c 



(5) 



Cytochrome b.^ (not bound) 



where the box expresses the whole enzyme, Y-LDH, consisting of two parts, 

 the dehydrogenase and the cytochrome b.2, moieties. Reaction (1) expresses a 

 dehydrogenase activity, and reaction (2) an electron transport in the 

 Y-LDH complex. If the externally added cytochrome c is reduced by 

 Y-LDH as fast as is the cytochrome h^ moiety, then reaction (2) proceeds 

 42 times faster than reaction (4). In this scheme of electron transport, the 

 intimate junction between the dehydrogenase and cytochrome b^ moieties 

 may be broken at the lower and higher pH ranges, and reaction (4) may take 

 the place of reaction (2). 



Modification of the Enzymic Activity of Y-LDH 



When Y-LDH is allowed to stand at pH 7-0 and 4-5''C in the absence of 

 the substrates, its enzymic activity gradually decreases. During storage, 



