IV. BIOCHEMICAL SYSTEMS 439 



s1u)\v(h1 no activity at all. Ochoa thinks that the reaction 



'riii;iiniiH' 4 '2 pliospliatc . ' 'riiiaiiiitic |)\ r(i|)li(»S|ili;il c 



is ivxiMsihlc. ( )xyji;en is essential for a reaction to the rijj;ht. The optiniuni 

 pll is about 8.5. ( loodhart and Sinclair,"** workinji; with l)loocl from avitamin- 

 ous pigeons, demonstrated that the nucleated blood cells can phosphorylate 

 thiamine. 



I). Siliprandi and X. Siliprandi^'*'' demonstrated that in rats and also in 

 human beings^**'' insulin is essential in the conversion of thiamine to cocar- 

 l)oxylase. 



A cell-free protein preparation containing the apocarboxylase has been 

 made from yeast (Weil-Malherbe^^) . He was able to demonstrate that 

 phosphorylation of thiamine occurs only in the presence of adenylpyrophos- 

 phoric acid or such reactions as entail its intermediate formation. 



Leuthardt and Nielsen"'* succeeded in preparing a purified apoenzyme 

 for the transformation of thiamine into cocarboxylase. This apoenzyme 

 needs the addition of adenosinetriphosphate and magnesium ions. The 

 optimal pH of the purified enzyme was found to be between pH 6.8 and 

 pH 6.9. 



Phosphopyruvic acid can act as a phosphate donor for the synthesis of 

 cocarl)oxylase in the presence of catalytic amounts of adenylic acid or 

 adenylpyrophosphate. Thus a direct transfer of phosphate from adenyl- 

 pol>'phosphate is the mechanism for phosphorylating thiamine. These 

 results were confirmed by Lipton and Elvehjem^" and by Quastel and 

 Webley.** The synthesis does not take place via the monophosphate 

 (Weil-Malherbe^'), for the production of thiamine pyrophosphate goes faster 

 from thiamine itself than from its monophosphate. The sj^nthesis of co- 

 carboxylase is almost completely inhibited by 0.005 M iodoacetic acid, and 

 only slightly inhibited by 0.04 M sodium fluoride (Lipton and Elvehjem^O- 

 Westenbrink and his collaborators studied the synthesis of cocarboxylase 

 by living yeast cells (Westenbrink et al}-). By adding a large excess of thia- 

 mine to the yeast, much more thiamine pyrophosphate is synthesized than 

 can be bound to the apocarboxylase present in the yeast; it is probably 



" R. S. Goodhart and H. M. Sinclair, Biochem. J. 33, 1099 (1939). 



"»D. Siliprandi and N. Siliprandi, Nature 168, 422 (1951). 



"bD. Siliprandi and N. Siliprandi, Nutrition Revs. 10, 214 (1952). 



" H. Weil-Malherbe, Biochem. J. 33, 1997 (1939). 



"» F. Leuthardt and H. Nielsen, Helv. Chim. Acta 35, 1196 (1952). 



8« M. A. Lipton and C. A. Elvehjem, Nature 145, 226 (1940). 



*' M. A. Lipton and C. A. Elvehjem, Cold Spring Harbor Symposia Quant. Biol. 7, 



184 (1939). 

 '2 H. G. K. Westenbrink, K. P. S. Parv(5, and H. Veldman, Biochim. et Biophi/s. Acta 



1, 154 (1947). 



