Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography.
We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.
Cook
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Schafer-Mitchell
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Angle
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Stohs
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D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000032:analysis* / Q000201:enzymology; D006454:Hemoglobins / Q000032:analysis; D006461:Hemolysis; D006801:Humans; D008274:Magnesium / Q000032:analysis; D015636:Magnesium Chloride; D009708:Nucleotidases / Q000097:blood*; D011741:Pyrimidine Nucleosides / Q000032:analysis; D011743:Pyrimidines / Q000097:blood*; D013056:Spectrophotometry, Ultraviolet
D006454:Hemoglobins; D011741:Pyrimidine Nucleosides; D011743:Pyrimidines; D015636:Magnesium Chloride; D009708:Nucleotidases; C040296:deoxypyrimidine 5'-nucleotidase; D008274:Magnesium