Stimulation of erythrocyte transketolase by added thiamin diphosphate is pH dependent

Abstract

Transketolase (EC 2.2.1.1, TK) is the only thiamin-dependent enzyme found in mature human erythrocytes. In thiamin (vitamin B,) deficiency, erythrocyte transketolase activity is usually diminished and can be stimulated by addition of the cofactor, thiamin diphosphate (TPP) to the cell-free hemolysate. The added TPP apparently activates the apotransketolase which reaches significant amounts in thiamin deficiency. The extent to which erythrocyte transketolase can be stimulated by the addition of TPP in vitro is now widely used as a measure of thiamin insufficiency [l-5] and has been termed ‘the TPP effect’. A high TPP effect in the absence of thiamin deficiency might be attributed to a variant transketolase [6] having decreased affinity for TPP [7]. In this paper we document a methodological problem which can also lead to an artefactually high TPP effect. Measurement of the TPP effect requires assay of transketolase activity both with and without added TPP. The widely used transketolase assays fall into two principle classes, each utilizing ribose 5-phosphate and xylulose 5-phosphate as substrates, with the latter generated in situ from the former. One class of assay measures the product sedoheptulose 7-phosphate [l-3, 8-101 by a calorimetric method [ll]. The other class of assay measures the product glyceraldehyde 3-phosphate by enzymic coupling to NADH oxidation [12] which is followed in a spectrophotometer. Since many of the described assays utilize low concentrations of buffer and high concentrations of hemolysate, we have investigated the pH dependence of the TPP effect.