RADIOMETRIC ASSAY PROCEDURE FOR THIAMINE PYROPHOSPHOKINASE ACTIVITY

A radiometric assay for the determination of TPPK activity in purified enzyme preparations and in crude extracts as well is presented. The distinctive feature of this method is the measurement of 14C-TPP formed in the TPPK reaction from 14C-thiamine. The optimum conditions of the reaction (pH optimum, reaction time, protein amount, ATP concentration, MgSO4 concentration, and thiamine concentration) are described. The method may be used to determine the cleavage of TPP. 1 × 10-3 M ADP or AMP inhibits the TPP synthesis by about 50% in the presence of 1.2 × 10-2 M ATP. Creatine phosphate and creatine kinase as ATP regenerating system stimulate the formation of TPP in the presence and in the absence of ATP. © 1969.