I 



III. BIOCHEMICAL SYSTEM 603 



in Fig. 3. In contrast to the pigeon liver assay, the arsenolysis assay is 

 sharply specific for intact CoA. 



4. Other CoA Assay Systems 



Assay systems have recently been devised where CoA concentration may 

 be measured spectrophotometrically in CoA-dependent DPN-reducing 

 systems. Sanadi and Littlefield in Green's laboratory-'- ^^ found that DPN 

 reduction by ketoglutarate in a muscle extract is CoA-dependent, and this 

 system may be used for assay. Stadtman-* found that the acetaldehyde- 

 DPN reaction in extracts of Clostridium khiyveri is CoA-dependent, and 

 this system likewise represents a promising assay system. 



C. THE CHEMISTRY OF COENZYME A 

 1. Phosphate and Adenylic Acid Content 



Nearly pure preparations of CoA contain 1 mole of adenosine and 3 

 moles of phosphate per mole of pantothenic acid.^* Only 1 of the 3 moles 

 of phosphate is split off by phosphomonoesterases (prostate phosphatase).^'^ 

 The partially dephosphorylated product is easily rephosphorylated by 

 ATP through a kinase present in pigeon liver extract (Novelli-®). This 

 fragment, therefore, assays in pigeon liver acetylation assay as CoA but 

 is inactive in the arsenolysis assay. It may be reactivated for arsenolysis 

 by incubation with ATP in pigeon liver extract. The monoester-linked 

 phosphate is located at the ribose of adenosine (Baddiley^'^) . 



Kaplan discovered an enzyme which specifically attacks a nucleotides 

 (-phospho-2-), but not the b form (-phospho-3-).^* With this enzyme he 

 studied the position of the monoester-linked phosphate in CoA and found 

 that it corresponded to the b nucleotide series. Interestingly enough, the 

 monoester phosphate in TPN corresponds to the a series, according to his 

 analysis. 



If CoA is treated with an enzyme preparation from potato similar to 

 the nucleotide pyrophosphatase of Kornberg,^^ it is inactivated for arseno- 

 lysis, but not for crude pigeon liver assay .^^ The pH optimum for the 



22 H. Beinert, R. W. Van Korff, D. E. Green, D. A. Buyske, R. E. Handschuraacher, 

 H. Higgins, and F. M. Strong, /. Am. Chem. Soc. 74, 854 (1952). 



23 D. R. Sanadi and J. W. Littlefield, /. Biol. Chem. 193, 683 (1951). 



24 R. M. Burton and E. R. Stadtman, /. Biol. Chem. 202, 873 (1953). 



25 W. H. DeVries, W. M. Govier, J. S. Evans, J. D. Gregory, G. D. Novelli, M. 

 Soodak, and F. Lipmann, J. Am. Chem. Soc. 72, 4838 (1950). 



2* G. D. Novelli, in Phosphorus Metabolism, Vol. I, p. 414. Johns Hopkins Press, 



Baltimore, 1951. 

 " J. Baddiley and E. M. Thain, /. Chem. Soc. 1951, 3421. 



28 L. Shuster and N. O. Kaplan, Federation Proc. 11, 286 (1952). 



29 A. Kornberg and W. E. Pricer, Jr., /. Biol. Chem. 182, 763 (1950). 



