604 PANTOTHENIC ACID 



splitting of the pyrophosphate bridge in CoA, however, was found to be 

 different from that of the pyridine nucleotides, being 4.5 rather than 7.^® 

 No phosphate, however, may be liberated with this enzyme. If the nucleo- 

 tidase-treated preparation is exposed, subsequently, to phosphomonoester- 

 ase, then all three phosphates are split off and almost complete inactivation 

 for all assay systems results. In analogy to the results of Romberg on DPN 

 and, in particular, on TPN with a similar enzyme,^^ it is concluded that the 

 nucleotidase splits a pyrophosphate bridge between a phosphopantothenate 

 derivative and diphosphoadenosine. The phosphopantothenate derivative, 

 now identified as phosphopantothenylthioethanolamine,^" was found to be 

 resynthesized to CoA with ATP and pigeon liver enzyme.^^ 



Evidence for the pyrophosphate bridge is further offered by the complete 

 inactivation with intestinal phosphatase, which, by virtue of its phos- 

 phodiesterase activity, opens the bridge and dephosphorylates completely. 

 The same effect obtains as mentioned, by a combination of dinucleotidase 

 treatment and partial dephosphorylation.^^- ^- 



The nature of the phosphate attachment to pantothenic acid has recently 

 been settled definitely after pantothenate-2'-phosphate, pantothenate- 

 4'-phosphate, the diphosphate, and the cyclic phosphate had been syn- 

 thesized and made available for comparison.^^- ^^ Baddiley chromatograph- 

 ically identified a fragment of CoA obtained by cautious alkaline hydrolysis 

 with pantothenate-4'-phosphate. This observation had already made it 

 most likely that the pyrophosphate bridge links between the 5' position 

 on ribose and the 4' position on pantothenic acid.^^- **■ ^^ Recently, Bad- 

 diley and Thain^^ have synthesized pantethine-4'-phosphate. This was 

 established as a fragment of CoA by enzymatic synthesis to CoA in a 

 pigeon liver fraction isolated by Levintow and Novelli^^ which acts specif- 

 ically on phosphorylated pantethine.^^ 



Final proof for the structure outlined was obtained recently by Hoagland 

 and Novelli,^^" who synthesized dephospho-CoA from ATP and synthetic 

 pantetheine-4'-phosphate and showed equivalent liberation of pyrophos- 

 phate according to the following equation : 



30 E. E. Snell, G. M. Brown, V. J. Peters, J. A. Craig, E. L. Wittle, J. A. Moore, 

 V. M. McGlohon, and 0. D. Bird, /. Am. Chem. Soc. 72, 5349 (1950). 



31 W. M. Govier and A. J. Gibbons, Arch. Biochem. and Biophys. 32, 347 (1951). 



32 G. D. Novelli, J. D. Gregory, R. M. Flynn, and F. J. Schmetz, Jr., Federation Proc. 

 10, 229 (1951). 



33 T. E. King and F. M. Strong, /. Biol. Chem. 189, 315 (1951); 191, 515 (1951). 

 3* J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 246. 



35 J. Baddiley and E. M. Thain, J. Chem. Soc. 1951, 2253. 



36 J. Baddiley, E. M. Thain, G. D. Novelli, and F. Lipmann, Nature 171, 76 (1953). 

 36" M. B. Hoagland and G. D. Novelli, /. Biol. Chem. 207, 767 (1954). 



