COENZYMES DERIVED FROM B VITAMINS 193 



liver or heart muscle. 259 Intact coenzyme A produces a comparable 

 response with this organism. 258 The cleavage in the coenzyme catalyzed 

 by the liver enzyme produces a derivative, still phosphorylated, which is 

 equally active for Acetobacter suboxydans. Incubation of either the intact 

 coenzyme or its phosphorylated intermediate with phosphatases, however, 

 destroys the activity for this organism. 



The pantothenic acid conjugate (PAC) isolated from heart muscle was 

 inactive when tested in enzymatic acetylation systems. 259 It seems likely, 

 then, that it is identical with the phosphorylated intermediate derived 

 from coenzyme A by treatment with the liver enzyme and probably is 

 formed from coenzyme A by an autolytic process during its concentration 

 from tissues. A. suboxydans consequently cannot be used for the specific 

 microbiological assay for the intact coenzyme. 



At present the only method of differentiating coenzyme A from some 

 of its degradation products is by the use of enzyme analyses. By a com- 

 bination of assays, using Lactobacillus arabinosis, A. suboxydans, and 

 enzymatic acetylation, it should be possible to work out a differential 

 assay for coenzyme A, free pantothenic acid, and the two compounds of 

 intermediate complexity. The activity under various testing conditions 

 is summarized: 



Free pantothenic acid 



Coenzyme A 



Phosphorylated intermediate (PAC?) 



Phosphatase-treated coenzyme =•= 



± long incubation or high concentration required. 



An enzymatic determination of coenzyme A can be made either by 

 (1) following the acetylation of choline (using as an indicator a biological 

 response — muscle contraction) , 260 or (2) determining the rate of acetyla- 

 tion of aromatic amines. 249 As a routine method of analysis, the latter 

 is preferred because of its greater simplicity and accuracy. A detailed 

 description of this assay procedure has been published. 261 This method 

 can be conveniently adapted to laboratories equipped for microbiological 

 analyses. The preparation of a suitable apoenzyme is not a problem, since 

 crude liver extracts can be used. The coenzyme originally present in these 

 extracts is completely inactivated if the extract is allowed to stand for 

 four hours at room temperature. 



Occurrence. By use of the rate of acetylation method just described, 

 the coenzyme A content of a number of animal tissues and various plant 

 materials has been ascertained. 261 The pantothenic acid content of these 

 sources was simultaneously measured by the conventional microbiological 

 procedure. A comparison indicates that within cells pantothenic acid exists 



