428 THIAMINE 



acid pH is very firm. Washing with water and dialysis does not remove a 

 trace of thiamine pyrophosphate. At pH about 8, the dissociation becomes 

 total, so that by washing with an alkaline phosphate buffer the thiamine 

 pyrophosphate can readily be removed. Also at pH 4.6 the thiamine pyro- 

 phosphate may be split off (Stumpf et alr'^). One exception, however, has 

 been found in the enzyme that brings about the condensation of acetalde- 

 hyde (Green et al.~^), for the coenzyme is completely dissociated even in 

 neutral solutions (Stumpf^-). 



Stumpf assumes that the thiamine pyrophosphate enzymes can be classi- 

 fied into two general groups, depending on the relative degrees of dissocia- 

 tion. The first group, to which the majority of the known carboxylases 

 belong, is not dissociated between pH 4.6 and 7.8. In the second group the 

 enzyme is dissociated even in neutral solutions. 



Ochoa and Peters (Ochoa^^) found that the addition of thiamine to apo- 

 carboxylase in the form of alkali-washed yeast greatly enhanced the capac- 

 ity of this apoenzyme for the production of carbon dioxide after addition 

 of the coenzyme thiamine pyrophosphate and magnesium. The activation 

 was maximal with doses of thiamine twenty times larger than the thiamine 

 pyrophosphate. Instead of thiamine itself also the pyrimidine part of 

 the molecule 2-methyl- 4- aminopy rimidy 1- 5- methylaminodihydrochloride 

 could be used (Ochoa and Peters^^). This proved that the activation is not 

 caused by synthesis of the added thiamine to thiamine pyrophosphate. 

 This stimulation by thiamine of the resynthesis of thiamine pyrophosphate 

 was much greater in baker's than in brewer's yeast (Lipton and Elvehjem"). 

 Westenbrink and his collaborators succeeded in giving an explanation for 

 this activation (Westenbrink and van Dorp^^ and Westenbrink et al.-'') 

 They found a powerful phosphatase in yeast ; this phosphatase is capable of 

 hydrolyzing the phosphoric esters of thiamine in a very short time. The 

 thiamine does not actually stimulate the carboxylase system but only in- 

 hibits the phosphatase that destroys the carboxylase. 



When the coenzyme once is bound to the protein, thiamine pyrophos- 

 phate is resistant to this phosphatase (Westenbrink et al}^). This is in ac- 



20 P. K. Stumpf, K. Zarudnaya, and D. E. Green, J. Biol. Chem. 167, 817 (1947). 



21 D. E. Green, W. W. Westerfeld, B. Vennesland, and W. E. Knox, /. Biol. Chem. 145. 

 69 (1942). 



" P. K. Stumpf, /. Biol. Chem. 159, 529 (1945). 



23 S. Ochoa, Nature 141, 831 (1938). 



2^ S. Ochoa and R. A. Peters, Biochem. J. 32, 1501 (1938). 



25 M. A. Lipton and C. A. Elvehjem, J. Biol. Chem. 136, 637 (1940). 



26 H. G. K. Westenbrink and D. A. van Dorp, Nature 145, 465 (1940). 



2^ H. G. K. Westenbrink, D. A. van Dorp, M. Grubcr, and H. Voldman. Enzymologia 



9,73(1940). 

 28 H. G. K. Westenbrink, A. F. Willebrands, and C. E. Kamminga, Enzymolonia 9, 



228 (1940). 



