ENZYMES CONCERNED WITH DIGESTION OF LIPIDS 39 



in the hydrolysis of acetylcholine, reacts readily with water to regenerate 

 the enzyme; however, the reaction between water and the corresponding 

 phosphorylated enzyme, produced by the addition of TEPP, is manifestly 

 slow, but not immeasurably so. It was shown that hydrolysis, with the 

 simultaneous regeneration of the enzyme, could be effected if the excess 

 TEPP was removed by hydrolysis or dialysis. Since the acylated enzyme 

 reacts readily with hydroxylamine or choline, these compounds have also 

 been used as regenerating agents for the TEPP-inhibited enzyme. It is 

 postulated that the enzyme and alkyl phosphate interact on the basic 

 group in the site of esterase activity on the enzyme surface, so that this 

 position is phosphorylated. This phosphorylated enzyme would then react 

 with the nucleophilic reagents (hydroxylamine or choline), resulting in the 

 regeneration of the enzyme. 



Nachmansohn et al. 22i point out that there is a difference between the 

 kinetics of inhibition by the alkaloids and by DFP. At higher concen- 

 trations, DFP was found to be more effective than were the alkaloids, 

 while the opposite condition obtains at lower concentrations. Moreover, 

 Mackworth and Webb 230 reported that the inhibition by DFP, unlike that 

 by eserine, is progressive ; it is not affected by substrate concentration, and 

 it cannot be prolonged by dialysis. 



Myers 231 noted a marked difference in the effect of electrolytes on cholin- 

 esterase inhibition as affected by eserine and DFP. In the case of the 

 alkaloidal inhibitors such as eserine and prostigmine, the affinity for the 

 cholinesterase is decreased by increasing the concentration of electrolytes 

 in the medium. On the other hand, alterations in salt concentration fail to 

 affect the affinity of cholinesterases for DFP appreciably. 



Prostigmine loses its inhibiting power during the course of acetylcholine 

 cleavage. 232 Goldstein and Hamlish 233 reported that the cholinesterase 

 inhibitors, eserine and prostigmine, are destroyed enzymatically by human 

 serum, by a human plasma fraction highly purified with respect to cholin- 

 esterase, and by the same purified fraction partially denatured by heat. 

 The rate of destruction of the inhibitor parallels the uninhibited activity 

 toward acetylcholine in all three preparations. On the basis of these 

 findings, it is postulated that the inhibitors are destroyed by cholinesterase. 

 The ratio of the inhibitor turnover number to that of acetylcholine is 5.5 X 

 10 ~ 7 for eserine and 1.2 X 10~ 7 for prostigmine. These results confirm the 



230 J. F. Mackworth and E. C. Webb, Biochem. J., 42, 91-95 (1948). 



231 D. K. Myers, Arch. Biochem., 27, 341-347 (1950). 



232 E. H. Maier and H. G. Bamraer, Biochem. Z., 322, 85-105 (1951). 



233 A. Goldstein and R. E. Hamlish, Arch. Biochem., 35, 12-22 (1952). / Qy> 



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