Simultaneous high-performance liquid chromatographic assay of the activities of erythrocytic hypoxanthine-guanine phosphoribosyl transferase and purine nucleoside phosphorylase.
Hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) and purine nucleoside phosphorylase (PNPase) activities were simultaneously determined in erythrocyte lysates, using the reversed-phase mode of high-performance liquid chromatography. Reaction conditions were developed to provide zero-order kinetics for both enzymes. The activities of the individual enzymes were calculated after incubation of cell lysates with the PNPase substrate, inosine. After sufficient hypoxanthine had been formed to saturate the HGPRTase, the co-enzyme phosphoribosylpyrophosphate and co-factor magnesium (Mg2+) were added to the incubation medium. The enzyme activities were calculated by measurement of the decrease in the PNPase substrate, inosine, and the increase in the HGPRTase product, inosine-5'-monophosphate.
D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000097:blood*; D007291:Inosine Monophosphate / Q000032:analysis; D010430:Pentosyltransferases / Q000097:blood*; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood*; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D007291:Inosine Monophosphate; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase