FUNCTION 



and reducing agents, the rate of reduction of aneurine by sodium 

 dithionite and by hydrogen in presence of colloidal palladium or 

 platinum black being three times as fast as the rate of reduction of 

 cocarboxylase ; a similar relationship was observed for the rates of 

 re-oxidation of the two reduction products. The reduced cocarboxy- 

 lase had neither vitamin nor enzyme-component activity, nor had the 

 substance formed by re-oxidation by means of histidine and ferri- 

 protoporphyrin, thus confirming the later result of Stern and Melnick. 

 It was concluded, therefore, that cocarboxylase is an integral part of 

 the activating protein when acting as a component of an enzyme 

 system, and that it does not owe its activity to reversible oxidation 

 and reduction. 



This was confirmed by in vitro experiments with tissues from 

 avitaminous animals, in which it was found that the addition of 

 aneurine accelerated condensation reactions of pyruvate leading to 

 the synthesis of carbohydrate, a-ketoglutarate, citrate, acetoacetate 

 and succinate. All these reactions start as condensation reactions of 

 pyruvate and in all of them there is a step in which an oxidative 

 decarboxylation occurs. Thus in the synthesis of carbohydrate, 

 aneurine pyrophosphate may catalyse either the carboxylation of 

 phosphopyruvate to phospho-enoloxaloacetate or the decarboxylation 

 of this compoimd to phosphoenolpyruvate, and it is impossible to 

 determine whether aneurine is a component of a condensation enzyme 

 or of an oxidative decarboxylation enzyme. The increased stability 

 of aneurine to reduction and oxidation following phosphorylation 

 rather argues against the latter alternative. 



The addition of aneurine also increased the oxidation and utilisa- 

 tion of a-ketoglutarate by the tissues of avitaminous rats, but only 

 when the aneurine had previously been incubated with the tissue to 

 make its phosphorylation possible. It was therefore concluded that 

 the activating protein of a-ketoglutarate oxidase is, like that of 

 pyruvate oxidase, an aneurine pyro phosphate-protein . 



The possibility that aneurine might be an oxidation catalyst has 

 been considered by other workers, and O. Zima and R. R. Williams ®^ 

 suggested a mechanism different from that proposed by Lipmann. 

 They pointed out that aneurine chloride hydrochloride could only 

 exist in solutions far more strongly acid than living tissue, and sug- 

 gested that in the cell the vitamin probably exists as a hemichloride ; 

 this can be obtained from the chloride hydrochloride by treatment 

 with excess potassium chloride. Two well-defined crystalline sodium 

 salts were obtained from aneurine chloride hydrochloride by addition 

 of alkali ; the one, obtained with sodium ethoxide, was deep yellow 

 in colour and the other, obtained with strong aqueous sodiiun hydroxide 

 solution, was white. These were assigned the structures : 



lOI 



