3iS 



R. BERMAN, I. B. WILSON, D. NACHMANSOHN 



VOL. 12 (1953) 



assay based upon the glutathione extinction coefficient and the biological acetylcholine measurement; 



Time (min) 



fiM of acetylcholine formed jml 

 Bioassay Nitroprusside test 



30 

 60 



0-33 

 0.67 



0-34 

 0.65 



These data validate the appearance of mercaptan as a measure of acetylation. However, it must 

 be noted that there is a difference - not yet explained - between the two systems: In the full system 

 including transacetylase, acetylphosphate etc. the activity was about 3 times as high as in the tests 

 with choline acetylase alone. 



RESULTS 



Figs. I and 2 demonstrate the time course of the acetylation reaction for different 

 concentrations of the substrates choHne and acetyl Co A. The choline curves show very 



2.00r 





10 15 20 40 



Choline u. mol/ml 



iMg. I. Acetylation of choline by choline ace- 

 Ivlase as function of choline concentration. 

 The reaction mixture contained 140 jug en- 

 zyme and the following components in juM 

 per ml: Acetyl CoA 5, phosphate buffer pH 7, 

 yo, choline as indicated in the graph. Total 

 volume I ml, t = 3i°C. 



2.00 



^68 

 Ac CoA a mol/ml 



Fig. 2. Acetylation of choline by choline ace- 

 tylase as function of Acetyl CoA concentra- 

 tion. The reaction mixture contained 140 /tig 

 enzyme and the following components in fiM 

 per ml: choline 10, phosphate buffer pH 7, 

 90, acetyl CoA as indicated in tlie graph. 

 Total volume i ml, t = 31 "C. 



marked enzyme saturation corresponding to a Michaelis-Menten constant of about 

 2-io~3 M. The acetyl CoA curves on the other hand show far less saturation in the 

 concentration range utilized and the data correspond to a Michaelis-Menten constant 

 on the order of 5-io~^ M. The curves are reasonably linear in time; the moderate rate 

 decline at longer times being nearly accounted for by the decrease in the concentration 

 of acetyl CoA. 



Table I shows the reaction rate as a function of time and enzyme concentration. 

 Again the moderate deviation from linearity at high enzyme concentration corresponds 

 to a decrease in acetyl CoA as the reaction proceeds. 

 References p. 324. 



