A rapid and sensitive radiometric assay procedure for thiamine pyrophosphokinase activity using anion-exchange paper discs.

Abstract

Abstract A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent 1 enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d -glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable. The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [ 14 C]thiamine as substrate. After incubation with the enzyme in the presence of Mg 2+ -ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [ 14 C]thiamine and partial retention of [ 14 C]TPP. This is quantitatively measured using the liquid scintillation counting technique. A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).