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(-14)C-
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 radioactivity 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.