IV. BIOCHEMICAL SYSTEMS 35 



in activity thereafter. From data obtained by these workers on 10-minute 

 incubations it appeared that, if two enzyme systems with different pH 

 optima for their activities were involved, the over-all activity in whole 

 rat liver homogenates was the same for the three pH values employed. 

 Since Williams and his coworkers^^" were interested in developing an assay 

 system for total rat liver homogenates, they also studied the product in- 

 hibition of the choline oxidase system. It was noted that the oxidation of 

 betaine aldehyde to betaine occurred at a much slower rate than the oxi- 

 dation of choline alone and that betaine aldehyde apparently inhibited the 

 oxidation of choline. Although betaine aldehyde was a potent inhibitor of 

 choline oxidase, betaine had no effect upon the oxidation of choline. Further 

 experiments showed that for the first 10 to 12 minutes oxidation of choline 

 proceeded linearly and that, as betaine aldehyde concentrations increased, 

 the rate of choline oxidation gradually decreased. Therefore, the first 10- 

 minute interval can be used as a measure of choline oxidase activity without 

 interference from the inhibitory action of betaine aldehyde or its slower 

 rate of oxidation. The requirements of the system for added cytochrome c 

 and for diphosphopyridine nucleotide were investigated also, and it was 

 concluded that for adequate assay no additional cofactors were necessary. 

 On the basis of the above findings Williams et aU^^ described an assay system 

 for measuring choline oxidase activity in total liver extracts. Their data 

 showed that rat liver choline oxidase is remarkably constant for comparable 

 animals. Niacinamide strongly inhibited the activity of the oxidase system 

 under all conditions. Both niacinamide and diphosphopyridine nucleotide 

 inhibited the activity of the oxidase system in total liver extracts. 



Further studies by Williams ^^^ on the mechanism of the metabolism of 

 choline and betaine aldehyde have shown that the latter metabolite can be 

 converted to betaine either aerobically or anaerobically, whereas choline 

 oxidase appears to be an oxygen-requiring system. 



Early studies on the intracellular distribution of the components of the 

 choline oxidase system were made by Lan,^^"' ^^^ who failed to find any ap- 

 preciable amount of the oxidase activity in isolated nuclei of rat liver cells. 

 Kensler and Langemann found 78 % of the oxidase activity in the mitochon- 

 drial fraction of rat liver preparations and also demonstrated that the addi- 

 tion of succinate greatly reduced the oxidation of choline by tissue homoge- 

 nates, presumably because of saturation of proton and electron acceptors. ^^*' 

 ^^* Concentration of choline oxidase activity in mitochondria was confirmed 



151 J. N. Williams, Jr. Proc. Sue. Exptl. Biol. Med. 78, 202 (1951). 



1^2 T. H. Lan, J. Biol. Chem. 151, 171 (1943). 



1" T. H. Lan, Cancer Research 4, 37, 42 (1944). 



1" C. J. Kensler and H. Langemann, J. Biol. Chem. 192, 551 (1951). 



155 H. Langemann and C. J. Kensler, Federation Proc. 11, 366 (1952). 



