An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases.
The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.
D000255:Adenosine Triphosphate; D000818:Animals; D000906:Antibodies / Q000032:analysis; D001932:Brain Neoplasms / Q000201:enzymology; D002384:Catalysis; D002460:Cell Line; D003856:Deoxyuracil Nucleotides / Q000096:biosynthesis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D009703:Nucleoside-Phosphate Kinase / Q000032:analysis* / Q000276:immunology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis*; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D003856:Deoxyuracil Nucleotides; D007457:Iodine Radioisotopes; D000255:Adenosine Triphosphate; C007267:2'-deoxyuridylic acid; D010770:Phosphotransferases; D013937:Thymidine Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase