Enzymes 211 



any residual activity toward acetylthiocholine after pretreat- 

 ment with DIPFP must be attributed to the "true" enzyme. 

 Actually, in view of certain discrepancies, it is, questionable 

 whether this simple scheme can be accepted at face value. 

 The hydrolysis of acetylthiocholine by liver is inhibited by 

 DIPFP only to the extent of 70 per cent; this is contrary to 

 the general consensus and the authors' own statement that 

 the hepatic enzyme is exclusively of the nonspecific type. 



Thiocholine, its acetate, and butyrate are available com- 

 mercially.^"^ 



The method recommended is a simplification of the 

 amended technique.^ 



124 



Method 



Make up the following stock solution: 



Copper sulfate (CUSO4 • 5H2O) 0.3 g. ^^ i 



Glycine 0.375 g. 



Magnesium chloride (MgCls • 6H2O) .... 1.0 g. 



Maleic acid 1.75 g. 



N (4 per cent) NaOH 30 ml. 



Hot saturated (about 40 per cent) solu- 

 tion of Na2S04 170 ml. 



This solution will keep indefinitely, although some of the 

 Na2S04 may crystallize out on cooling. Its pH is around 6. 



For use, dissolve about 20 mg. of acetylthiocholine iodide ^ 

 in fev/ drops of water and add 10 ml. of the stock solution. 

 Incubate teased tissues or fresh-frozen sections for 10-60 

 minutes at 37° C. Rinse them in two or three changes of 

 saturated Na2S04 and immerse them in a dilute solution of 

 yellow ammonium sulfide. Counterstain as desired, dehy- 

 drate, and mount. Sites of activity are stained dark brown 

 ( shade of cupric sulfide ) . 



If DIPFP is used as an inhibitor, the tissues are first 

 treated with a 10~^ M solution of the compound in saturated 



123. From LaWall and Harrison, 1921 Walnut St., Philadelphia 3, Pa. 



124. Koelle, G. B.: J. Pharmacol. & Exper. Therap., 103:153, 1951. 



