3l6 R. BERMAN, I. B. WILSON, D. NACHMANSOHN VOL. 12 (1953) 



The acetylating enzyme requires a coenzyme, later called Coenzyme A (CoA) , which 

 was discovered simultaneously and independently in three laboratories in 1945 (LiP- 

 MANN AND Kaplan^, Lipton in Barron's laboratory^, Nachmansohn and Berman^). 

 The structure of CoA is today well established by the work of various laboratories, es- 

 pecially by the work of Lipmann and his associates'-^. An essential progress was the isola- 

 tion of acetyl CoA ("active acetate") by Lynen and Reichert^ and their discovery that 

 this compound is an acetyl thioester. The thioester was postulated by Lynen to be a new 

 type of energy rich compound and this assumption was confirmed by the work of Stern, 

 OcHOA and Lynen^" and Stadtman^^. During the last few years the analysis of the 

 mechanism of acetylation has made rapid progress, mainly by the work of Lipmann, 

 OcHOA, Lynen, Green, Barker and Stadtman and their associates. 



The availability of acetyl CoA makes possible an investigation of the specificity of 

 choline acetylase, the enzyme which transfers the acetyl group from acetyl CoA to 

 choline^^. The molecular forces acting between the substrates and this enzyme appeared 

 of special interest in view ol the results obtained in studies of the molecular forces acting 

 between acet^dcholine and acetylcholinesterase-- i3-i5_ 



It is a great privilege and pleasure for the authors to present this first study on the 

 specific features of choline acetylase and their biological significance as a tribute to 

 Professor Otto WarburCx on the occasion of his 70th birthday. 



METHODS 



Purification 



Acetone-dried powder prepared from head ganglia of Squid^^ was extracted with 0.05 .1/ 

 K2HPO4 buffer of pH 7.4 and was centrifuged in the Spinco preparatory ultracentrifuge. The pro 

 teins of the supernatant solution were precipitated with an equal volume of 50 % ammonium sulfate 

 and centrifuged. The precipitate was dissolved in o.i M KgHPO^ buffer of pH 7.8. The ammonium 

 sulfate was removed by dialysis against 0.05 M KoHPOj buffer of pH 7.4. Protamine sulfate, in a 2 % 

 solution was added (i mg per 10 mg protein) and the precipitate formed was removed by centrifuga- 

 tion.When large amounts of materials were used, an ammonium sulfate fractionation was carried out 

 at this stage and the protein fractionated between 16 and 28 %. With smaller amounts the next step 

 was carried out directly, consisting of treatment with 3.3 mg of calcium PO^ gel per mg of protein at 

 pH 6.2. The gel was centrifuged and eluted with 0.05 M phosphate buffer of pH 7.5 and the eluate 

 discarded. Futher elution with 0.2 M phosphate buffer of pH 8.2 contained most of the enzyme. The 

 eluate was again fractionated with ammonium sulfate, between 16 and 32 %. 



The data of one preparation are given as example; although in this way only a lo-fold jnirifica- 

 tion was obtained, the enzyme solution at this stage has a satisfactory stability and is adequate 

 for the analysis desired, especially the deacylase activity present in the crude preparation is removed 

 in this process. 



Assay methods during purification 



The enzyme activity was tested by assaying the acetylcholine formed with the method of 

 Hestrin^^. The preparations used were the same as described in a previous papcr^-, except for CoA. 

 The CoA preparation was obtained from Pabst and was about 75% pure. The reaction mixture con- 

 tained the following components in micromoles per ml: choline chloride 50, MgClg 5, tetraethyl pyro- 



* fiM ACh formed pvr mg ])rotein per hour. 

 References p. 324. 



