Bakers'' Yeast Lactate Dehydrogenase 



555 



the ability to reduce methylene blue, ferricyanide and cytochrome c decreases 

 much faster than does the abihty to reduce phenazine methosulphate. 

 Figure 1 shows a notable difference in the enzymic activities between the 

 original enzyme and the enzyme stored for 72 hr (Horio, Yamasliita and 

 Okunuki, 1959). Such partial modification of Y-LDH can be demonstrated 

 within a much shorter time if the storage is carried out at pH 5-0, at which 

 pH it has been suggested {vide supra) that the electron-transferring pathway 



5 10 15 



ENZYME CONCENTRATION IN UNITS 



Fig. 1. Partial modification of bakers' yeast lactate dehydrogenase. The enzymic 

 activities of the original and treated Y-LDH were assayed by two different 

 methods: phenazine methosulphate method (curves, Opu and Mp^), and 

 methylene blue method (curves, Omb and Mmb)- Reduction of methylene blue 

 by lactate was carried out in Thunberg tubes having an oxygen-free nitrogen gas 

 phase at 35°C. The enzymic activities were compared between freshly-prepared 

 Y-LDH (curves, OpM and Omb) and a sample of the Y-LDH stored in the 

 absence of lactate at pH 70, and at 4-5°C, for 72 hr. One unit of Y-LDH 

 (original and treated) was defined as the quantity of the enzyme capable of 

 consuming 20 //I. of oxygen in the presence of phenazine methosulphate and 

 lactate in the first 5 min at 38°C. 



in Y-LDH might be most easily damaged between the dehydrogenase and 

 cytochrome b^ moiety. If lactate is aerobically added to the modified Y-LDH, 

 the modified enzyme shows the reduced absorption spectrum of cytochrome 

 b.2 only slightly or not at all, even at concentrations at which the original 

 and modified enzymes show the same enzymic activity by the phenazine 

 methosulphate method, and at which the dithionite-reduced absorption 

 spectra of both samples are easily measurable. The modified enzyme has 

 not yet been purified to a state free of cytochrome b^. 



Protection by Substrates against the Inactivation of Y-LDH by Heat 



Bach et al. (1946) found that lactate remarkably protects Y-LDH against 

 heat-denaturation, and they adopt this property to their Y-LDH purification 

 procedure. The other substrates can protect against denaturation, although 

 they are inferior to lactate in their protective ability. The protective abilities 

 of the various substrates vary in the same manner as do the rates at which 



