64 INFLUENCE OF TEMPERATURE ON BIOLOGICAL SYSTEMS 



HEAT-LABILE INHIBITORS 



Nature of Enzyme-Inhibitor Complex. As has been mentioned, the 

 basis for this apparent 'heat activation' resides in the fortuitous interac- 

 tion of a heat-labile inhibitor and a heat-stable enzyme. Some extracts of 

 Proteus vulgaris contain roughly a 50% excess of free inhibitor, that is, 

 inhibitor not already tightly bound to the enzyme. The effect of this free 

 inhibitor on the active purified DPN pyrophosphatase can be seen in 

 figure 2. Preincubation of enzyme and inhibitor would probably have made 

 the linear relationship between the amount of inhibitor and the degree of 

 inhibition evident earlier in the time course. 



The inhibitor is non-dialyzable and has been purified several fold by 

 alkaline ammonium sulfate and ethanol fractionation. The purified en- 

 zyme-inhibitor complex contains no detectable ribonucleic acid, and this 

 inactive complex is not activated by ribonuclease. Neither trypsin nor 

 pepsin causes discernible activation of the complex. Acid treatment (pH 

 1-2) for 10 minutes at 25°C destroys the free inhibitor. Similar treatment 

 inactivates bound inhibitor and in this fashion the enzyme is converted 

 to the fully active form without heating. The enzyme remains active even 

 after the pH is restored to neutrality. The above information suggests that 

 the inhibitor is a protein and that it is probably bound to the enzyme by 

 an ionic linkage rather than by a peptide or similarly strong bond. 



Working with Mycobacterium which contains roughly a 12-fold excess 

 of inhibitor over enzyme, Kern (7) has been able to purify the protein 

 inhibitor of DPNase quite extensively. Using this preparation he has been 

 able to show that the inhibitor forms an essentially undissociable complex 

 at neutral pH with the enzyme and that the formation of this complex- 

 occurs at a measurable rate. As with the Proteus enzyme, acid treatment 

 will activate the inhibited enzyme. Unlike the Proteus enzyme, however, 

 restoring the pH to neutrality will return the enzyme to the inhibited 

 form. Then, only after boiling is DPNase activity once again found. Thus, 

 like the trypsin-pancreatic inhibitor compound (10) this DPNase-inhibi- 

 tor complex is reversibly dissociable at acid pH (7). 



Heat-lability of Purified Inhibitor. The heat lability of the protein 

 inhibitors of the Proteus enzyme and the Mycobacterium enzyme is 

 rather different. Kern (7) has shown that while the inhibitor in Myco- 

 bacterium is markedly heat-labile in crude extracts, the purified in- 

 hibitor is surprisingly heat-stable. The heat lability in crude extracts is 

 increased by NaCl but the purified inhibitor remains stable even in the 

 presence of salts. The factor which contributes to the heat lability of this 

 inhibitor in crude extracts of Mycobacterium is not as yet known. In con- 

 trast, the inhibitor from Proteus remains heat-labile even after many steps 

 of purification. 



