50 



1, lODOACETATE AND lODOACETAMIDE 



that the discrepancy between their results and those of Dixon (1937) was 

 due to different times of incubation. Certain enzymes, such as myosin 

 ATPase (Bailey and Perry, 1947) and aspartase (EUfolk, 1953), require 

 many hours; the latter enzyme is inhibited 20% at 8 hr and 50% at 24 hr 

 by 10-50 mM iodoacetamide. If the enzyme is not preincubated with inhib- 

 itor the results mean very little, and probably many of the low inhibitions 

 observed are due to a failure to appreciate the time factor. Even those en- 

 zymes which react relatively rapidly with iodoacetate require some pre- 

 incubation; thus, 3-PGDH from Escherichia coli is inhibited significantly 

 more after 10 min preincubation than without preincubation (Still, 1940). 

 It is very difficult to generalize but, for practical purposes and taking into 

 account the usual period over which most cellular studies of glycolysis are 

 carried out, one might say that at least a 30-min preincubation period should 

 be used when testing an enzyme for its response to iodoacetate. Very few 

 enzymes react completely with reasonable concentrations of iodoacetate 

 within 30 min. The results with rice alcohol dehydrogenase (Fig. 1-6), are 



Fig. 1-6. Rates of inhibition of rice alcohol 

 dehydrogenase by iodoacetate at different con- 

 centrations. (Data from App and Meiss, 1958.) 



probably quite typical, and emphasize how meaningless reported degrees 

 of inhibition may be without accurate statements of the times of contact 

 of the enzyme with inhibitor. Yet in over half the studies now being pub- 

 lished, it is impossible to find mention of preincubation procedures. 



