68 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



withstand boiling requires a dialyzable factor. Extracts dialyzed for 18 

 hours against distilled water are completely devoid of activity after boil- 

 ing. As can be seen in figure 5, when inorganic pyrophosphate is added to 

 the dialyzed preparation prior to boiling, essentially full activity is re- 

 tained. Cobalt is added to the reaction mixtures since this metal or manga- 

 nese is needed for enzymatic activity and is apparently lost on dialysis. 

 An as yet unidentified organic phosphate compound in the barium-soluble, 

 alcohol-insoluble fraction of trichloracetic acid extracts of fresh Proteus 

 cells will also afford protection against high temperatures. DPN, NMN, 

 5'-adenylic acid (5'- AMP), metaphosphate, 5'-inosinic acid (5'-IMP), 

 TPN, CoA, ATP and ribose-5-phosphate will not replace either this com- 

 pound or inorganic pyrophosphate. The concentration of inorganic pyro- 

 phosphate in Proteus extracts is far below the level that affords maximal 

 protection. Thus, it would not appear to be the 'natural' protective factor. 

 The protection afforded by pyrophosphate applies to the purified enzyme 



Table 2. Purified proteus enzyme: protection against 



BOILING BY NA PYROPHOSPHATE 



NA PYROPHOSPHATE CONC. % PROTECTION 



1 X 10-1 81 



6.4 X 10-2 59 



6.4 X 10-=' 53 



1 X 10-^ 10 



1 X 10-^ 



as well as to crude extracts. In table 2 it can be seen that a high degree of 

 protection is conferred on the purified enzyme by sodium pyrophosphate. 

 It is interesting that the concentration of the latter required is about 18 

 times the optimum concentration for protection of the crude enzyme. A 

 concentration of pyrophosphate similar to that used with the crude ex- 

 tracts will protect the enzyme when the purified enzyme-inhibitor complex 

 is boiled. The explanation of this is not very clear although it might sug- 

 gest that the presence of the inhibitor, although unable to protect by it- 

 self, might potentiate the effect of pyrophosphate. 



A similar protective factor for the Proteus enzyme has been found in the 

 supernatant fraction of boiled serum. Liver and yeast extracts do not ap- 

 pear to contain this factor in appreciable amounts. 



Previously it had been noted that extracts of Proteus vulgaris con- 

 tained a second 'heat-activated' enzyme, a 5' nucleotidase. It has been 

 difficult to separate these two enzymes by protein fractionation procedures. 

 However, they appear to be distinct enzymes. The pyrophosphatase has an 

 absolute requirement for either cobalt or manganese whereas the 5' nucleo- 

 tidase has no metal requirement. Thus far the inhibitor preparations have 



