The manometric determination of thiamine pyrophosphate and the inhibition of the acid yeast phosphatase

Abstract

Sodium molybdate is a powerful inhibitor of the acid yeast phosphatase in both fresh baker's yeast and dried brewer's yeast, provided that the yeast is suspended in a suitable buffer. It displays no action in citrate or phosphate buffers, but is active in acetate or maleate buffers, both at the optimum pH of 3·7 of the phosphatase and at pH 6.2. Depending upon the relative concentrations of yeast and phosphate ester substrate, either 0.1 m M or 1 m M molybdate gives adequate protection against hydrolysis. In the manometric determination of thiamine pyrophosphate molybdate can replace thiamine as inhibitor of acid phosphatase with full retention of sensitivity if the assay is performed in 0.1 M maleate (pH 6.2) instead of 0.1 M phosphate (pH 6.2). In studies of the phosphorylation of thiamine and its antagonists by ATP molybdate has also proved to be useful for inhibiting the acid phosphatase present in preparations of yeast thiaminokinase.