M. N. SWARTZ, N. O. KAPLAN AND M. E. FRECH 



65 



The requirement for high temperatures for complete activation of the 

 Proteus enzyme can be seen in figure 3. Inhibited enzyme preparations 

 (enzyme-inhibitor complexes) were heated for two minutes at various 

 temperatures and DPN pyrophosphatase activity was then assayed at 37°. 

 Maximum activity was achieved only after boiling the preparations. 



Specificity of Inhibitors. The protein inhibitors obtained from the 

 various bacteria previously mentioned appear to be quite species-specific 

 and there has been no evidence of cross reaction thus far. The inhibitor 

 from Mycobacterium butyricum does not inhibit the DPNase from pig 

 brain, Neuros-pora crassa, Bacillus subtilis or Chromobacter violaceum (7) . 



100 



Fig. 3. Heat activation of in- 

 hibited DPN pyrophosphatase. 

 The enzyme preparation was a 

 1-6 sonicate of Proteus vulgaris 

 cells in water. DPN splitting 

 was measured by alcohol dehy- 

 drogenase method. 



>- 

 H 



> 



1- 

 O 

 < 



O 



cr 

 u 



Ol 



20 40 60 

 TEMPERATURE 



80 



100 



Also, the DPN pyrophosphatase of Proteus vulgaris is not affected by the 

 inhibitor from Mycobacterium. 



Further information concerning the nature of the enzyme-inhibitor com- 

 plex may be gleaned from some studies in which antibodies to the in- 

 hibited enzyme of Proteus vulgaris were produced in rabbits.^ These anti- 

 bodies significantly inhibited purified active DPN pyrophosphatase 

 preparations from the same organism. It is quite likely that the complex 

 dissociated after injection or that the inhibitor was destroyed, exposing 

 the 'active' and antigenic site on the enzyme. Alternatively, it is possible 

 that the antigenic site and 'active' site on the enzyme are contiguous. It 

 is interesting to note that the antibodies to the heat-stable enzyme are 

 themselves heat-labile, i.e. boiling the purified enzyme inhibited by anti- 

 body will restore activity. Just as in the case of the inhibitors there ap- 



^The work with antibodies was done in collaboration with Dr. A. G. Osier, De- 

 partment of Microbiology, School of Public Health and Hygiene. 



