Various Forms of Yeast Lactate Dehydrogenase 545 



Lactate Dehydrogenase If (LHD II) 



This was eluted at pH 5-3 with 0-04 m NaCl. The enzyme was not eluted 

 as a distinct protein peak, as the fraction was contaminated by (partly 

 dialysable) polypeptides and also polynucleotides. Proteolytic and nucleolytic 

 degradation took place during the elution procedure. Flavin and haem were 

 eluted as one distinct peak. £"280/^260 reached a minimum with this peak, thus 

 indicating affinity of the enzyme for the polynucleotides. The purest pre- 

 paration contained one haem/97,000 g of protein, and was thus as pure as 

 the crystalline enzyme. After dialysis, the enzyme still contained 2-3% of 

 polynucleotides. These were all of the ribose type. The crystalline enzyme 

 contains deoxyribonucleotide (Appleby, 1957; see Morton, 1958). Ribo- 

 nuclease and deoxyribonuclease did not affect fraction II, just as they did 

 not inactivate the crystaUine enzyme (Appleby and Morton, 1955, 1960). 

 Between LDH II and III, cytochrome c peroxidase was eluted as a brown zone 

 with one haem/200,000 g of protein. 



Some preparations of this material underwent inactivation suddenly (in 

 the course of minutes) when held at 0°C. This was frequently accompanied 

 by the formation of a colourless protein precipitate. This precipitate, which 

 contained much of the nucleotides, may be a part of the enzyme protein. 

 One haem/68,000 g of polypeptides remained dissolved. The inactivation 

 was probably due to proteolytic enzymes, which were present even in the best 

 preparations. Crystalline trypsin and chymotrypsin increased the rate of 

 inactivation. 



Inactivation was associated with the appearance of fluorescence (see 

 Appleby and Morton, 1954). The rate of dissociation of the flavin was 

 increased by sodium chloride in the acidic range (around pH 5), thus sug- 

 gesting that ionic bonds were involved (Theorell and Nygaard, 1954). The 

 dissociation was irreversible. />-Chloromercuribenzoate (10 * m) caused 

 fluorescence to appear immediately. 



REACTION PROPERTIES 

 Crude Extracts 



When the acetone precipitate (cf. Nygaard, 1958, 1959a, b) (dissolved in 

 water or in phosphate buffer at pH 7) was incubated at 55-57°C for 5 min. 

 or treated with /7-chloromercuribenzoate (pCMB, 3 x 10^ m), 90% or more 

 of the LDH activity was destroyed. The properties of the remaining enzyme 

 were quite different from those of the original solution. In the first place, the 

 ratio of the reduction of cytochrome c to the reduction of ferricyanide and 

 2:6-dichlorophenolindophenol was high for the stable enzyme. Secondly, 

 the reaction of the stable enzyme with cytochrome c was much more aftected 

 by changes in the salt concentration. Figure 1 illustrates the reaction properties 

 of a crude preparation which contained 10% of heat-stable enzyme. It is 



