Optimized thymidylate kinase assay, based on enzymatically synthesized 5-(/sup 125/I)iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

Journal Article · Fri May 01 04:00:00 EDT 1987 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(87)90426-X· OSTI ID:6326611

Karlstroem, A R.G.; Gronowitz, J S

The biological synthesis and purification of 5-(/sup 125/I)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.

• Research Organization: Uppsala Univ., Sweden
• OSTI ID: 6326611
• Journal Information: Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 162:2; ISSN ANBCA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIBODIES
BETA DECAY RADIOISOTOPES
BIOSYNTHESIS
CATALYSIS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DISEASES
ELECTRON CAPTURE RADIOISOTOPES
ENZYME ACTIVITY
ENZYME IMMUNOASSAY
ENZYMES
FRACTIONATION
HERPES SIMPLEX
IMMUNOASSAY
INFECTIOUS DISEASES
INHIBITION
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
RADIOISOTOPES
SEPARATION PROCESSES
SKIN DISEASES
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
UMP
VIRAL DISEASES