310 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 4 (1950) 



ACYLATION REACTIONS MEDIATED BY PURIFIED ACETYLCHOLINE 



ESTERASE 11* 



by 



SHLOMO HESTRIN** 



Department of Neurology, College of Physicians and Surgeons, Columbia University, 



New York (U.S.A.) 



The probability that acetylchoUne esterase plays a role in the generation of the 

 action potential^ lends special interest to the study of the nature of this enzyme and of 

 the reactions which it may mediate. In an earlier communication^ the ability of the 

 electric tissue esterase of Electrophorus electricus to mediate acylations of choline and 

 hydroxylamine was noted. In the present report, factors which govern the rate and 

 extent of these reactions are considered. 



The specificity and affinity of purified electric tissues esterase for a wide range of 

 substrates and inhibitors have been studied by Nachmansohn et al.^> ^ and more 

 recently by Augustinsson^'^. An important function of the enzyme — the hydrolysis 

 of esters as a function of p^ —has not been described previously. The manometric method 

 of esterase assay is conveniently applicable within a narrow range of p^- Characterization 

 of the Ph function of the enzyme by the potentiometric technique for the determination 

 of the acid reaction product would be feasible but laborious. A colorimetric method' for 

 the assay of ester in the presence of excess of products of ester hydrolysis affords a 

 convenient procedure for assay of esterase activity at any desired p^. The method is 

 applicable equally to measurement of both hydrolysis and synthesis of the ester and 

 with its aid information concerning the p^ function of an esterase is easily obtainable. 



METHODS 



Acetycholine and propionylcholine were determined according to the procedure previously 

 described'. Aliquots of 0.5 or i.o ml of the test solution containing 0.3 to 4.0 jjM. of ester were used 

 for the determinations. 



Acethyldroxamic and propionhydroxamic acid were measured in aliquots of 0.5 or o.i ml con- 

 taining 0.3 to 4.0 /iM. The samples were brought to pn i. 0-1.4 with hydrochloric acid and then esti- 

 mated colorimetrically with i °/^^ ferric chloride essentially as in the method for the determination of 

 acetylcholine'. 



The Klett photoelectric colorimeter was used with green filter 54. 



Enzyme 



Acetylcholine esterase of the electric tissue of Electrophorus electricus was used. The enzyme was 

 purified according to the method described by Rothenberg and Nachmansohn*. The enzyme was 

 dissolved in a medium of sodium pho.sphate 0.05 M, magnesium chloride 0.02 M, and sodium chloride 

 0.1 M at ph 7-0 and stored in the cold at 4^^ C. Stock enzyme solutions were diluted into 2.8% gelatin 

 freshly before use. In the hydrolysis experiments the final dilution of the enzyme solution was in the 

 order of magnitude of one part in ten thousand ; in the experiments on acylation a much higher enzyme 

 concentration — an order of magnitude of one part in ten — was used. 



* This work has been carried out under grants from the U. S. Public Health Service and the 

 Office rf Naval Research. 



Present address: The Hebrew University, Jerusalem, Israel. 



References p. 321. 



