36 CHOLINE 



in extensive studies by Williams. ^^^ A potent heat labile inhibitor of choline 

 oxidase was found in the supernatant fraction. These studies also showed 

 that the betaine aldehyde oxidase activity of the supernatant fraction can 

 be considerably increased if diphosphopyridine nucleotide is added. The 

 nuclei and microsomes are unaffected by addition of this nucleotide. 



Although choline oxidase activity has been found in the livers and kidneys 

 of rats, chicks, rabbits, dogs, and monkeys,^" most of the in vitro studies 

 on this enzyme system have used tissues of the rat. It has been shown that 

 choline oxidase in the rat liver is cyanide-sensitive and that it is readily 

 inhibited by copper and phenylhydrazine.^** Barron and Singer^^^ have 

 classified it in the group of sulfhydryl enzymes. Choline oxidase loses its 

 activity at a fairly rapid rate if partially purified. The rate of loss, which is 

 that of a first-order reaction, is more rapid at pH 6.7 than at 7.8. Cystine 

 and semicarbazide increase the rate of inactivation both in presence and 

 absence of choline. Nickel, cobalt, and iron salts not only increase the in- 

 activation rate of the enzyme but also combine reversibly with a group on 

 the enzyme to inhibit it.^^" Halogenated alkylamines are powerful inhibitors 

 for choline oxidase.'" Among these is the nitrogen mustard, methylbis- 

 (/3-chloroethyl) amine, which irreversibly inhibits choline oxidase. Colter 

 and QuasteP''- observed that benzedrine, ephedrine, tyramine, methylamine, 

 aminoethanol, and histamine not only reversibly inhibit choline oxidase 

 but also prevent the irreversible inhibition by the nitrogen mustard. 



The enzyme in rat liver which oxidizes choline was named choline de- 

 hydrogenase by Bargoni and Di Bella. '^^ Its activity was ascribed to free 

 amino and carbonyl rather than to mercapto groups. The enzyme shows 

 no color with nitroprusside nor with Grote's reagent and is inhibited by 

 cyanide but not by malonate. The dehydrogenation system involves the 

 oxidation of reduced cytochrome c. 



At pH values below 7.0, stearic, palmitic, and oleic acids at 0.004 M 

 concentrations inhibit choline oxidase activity 61 %, 41 %, and 29 %, respec- 

 tively. Fats and mixtures of cephaline and lecithin are without effect. It 

 has been suggested'^^ that the fatty acids in liver may determine to some 

 degree how much free choline can be oxidized and how much can be used 

 for synthesis of phospholipids. 



5« J. N. Williams, Jr.. /. Biol. Chem. 194, 139 (1952); 195, 37 (1952); 197, 709 (1952). 

 " J. S. Dinning, C. K. Keith, and P. L. Day, Arch. Biochem. 24, 463 (1949). 



58 F. Bernheim, /. Biol. Chem. 133, 485 (1940). 



59 E. S. G. Barron and T. P. Singer, Science 97, 356 (1943). 



60 G. S. Eadie and F. Bernheim, J. Biol. Chem. 185, 731 (1950). 



" E. S. G. Barron, G. R. Bartlett, and Z. B. Miller, ./. Exptl. Med. 87, 489 (1948). 



62 J. S. Colter and J. H. Quastel, Nature 166, 773 (1950). 



63 N. Bargoni and S. Di Bella, Atti accad. sd. Torino 84, 149 (1949-1950). 



64 F. Bernheim, /. Biol. Chem. 133, 291 (1940). 



