Active Hypoxanthine Phosphoribosyltransferase 1 (HPRT1)

Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) is an enzyme central to the purine salvage pathway. It utilizes phosphoribosylpyrophosphate (PRPP) to transform hypoxanthine into inosine monophosphate (IMP) and guanine into guanosine monophosphate (GMP). APRT, on the other hand, catalyzes the conversion of adenine to adenosine monophosphate (AMP) also with PRPP. When HPRT1 and APRT interact, they can potentially co - ordinate the utilization of PRPP.Thus a functional ELISA assay was conducted to detect the interaction of recombinant human HPRT1 and recombinant human APRT. Briefly, HPRT1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to APRT-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HPRT1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant human HPRT1 and recombinant human APRT was shown in Figure 1, the EC50 for this effect is 0.092ug/mL.