Synthesis and purification of [γP32]-ATP

The synthesis of [γP32]-ATP has been carried out using an enzymes procedure. The compound was formed by the phosphorylation of ADP during the enzymatic conversion of L-α-glycerol-phosphate to 3-phosphoglycerate. In the present study, lactatedehydrogenase and sodium pyruvat were used in order to maintain β-NAD+ concentration and to push the reaction of glyceralaldehyde-3-phosphate dehydrogenase towards the formation of 1,3-diphosphoglycerate. L-α-glycerolphosphate was used as primary substrate, as it is more stable than DL-glyceraldehyde-3-phosphate. The enzymatic reaction was stopped by immersing the reaction vessel in boiling water for about 10 minutes. The labelled [γP32]-ATP formed was separated by thin layer chromatography using PEI-cellulose and the spots of [γP32]-ATP and inorganic P32 residue located by autoradiography using X-ray film. The optimum time for the reaction at room temperature was 90 minutes with a labeling efficiency of 94.9 %. Purification of the [γP32]-ATP by anion exchange chromatography using DEAE sephadex yielded a purity of more than 95%. The results showed that the labeled compound [γP32]-ATP can be synthesized via an enzymatic process with a satisfactory yield. (author), 4 refs, 2 tabs, 2 figs