Studies on ATP

The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2-¹⁴C-thiazole]thiamine was used for the synthesis of [¹⁴C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates,¹⁴C radio-activity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [¹⁴C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes.