Important note: Information in this article was accurate in 1990. The state of the art may have changed since the publication date.
SUBSTRATE ANALOGS FOR DETECTION CHARACTERIZATION AND INHIBITION STUDIES OF ENZYMES ASSOCIATED WITH THE DNA REPLICATION OF VIRUSES AND CELLS
Diss Abstr Int [C]; 50(4):682 1989. Unique Identifier : AIDSLINE ICDB/90662079 Karlstrom AR; Uppsala Universitet, Sweden
Abstract:
The biological synthesis of 5-[125I] iododeoxyuridine-monophosphate and triphosphate by enzymatic phosphorylation of 5-[125I] iododeoxyuridine and their subsequent purifications are described. The specificity and gain in sensitivity achieved with these substrates in enzyme assays is demonstrated. The increased sensitivities were exploited for the detection of viral enzyme activity-blocking antibodies (ab) in human sera. From studies on the immunological relationship between the TMPK and thymidine kinase (TK) induced by herpes simplex virus (HSV)-1 and HSV-2, it was concluded that the HSV-2, but not the HSV-1, TMPK and TK activities are mediated by different catalytic sites. The increased sensitivity of the reverse transcriptase (RT) assay made direct detection of human immunodeficiency virus (HIV)RT ab in patients possible. All sera from HIV infected individuals were found positive; however, the earlier reported relation between HIV RT ab amount and clinical status could not be verified. The improved measurement of TMPK for analysis of enzyme activity in tumor biopsies, as well as the use of the new DNA assay for serum analyses, is illustrated. The different forms of TK and DNAp found in sera from patients with leukemia, B12-deficiency and human cytomegalovirus infections were characterized with respect to mol wt. The major TK form found in serum had a mol wt of 730 kD which differed from that found in cells (58 kD). The large 730 kD form was shown to be quantitatively converted into the smaller 58 kD form in the presence of dithioerythriol (DTE) and exhibited the same kinetic behavior as the proliferation associated TK1 found in HeLa cells. The DNAp had an mol wt of 240 kD which was to converted into a smaller form (56 kD) in the presence of DTE. It was concluded that both TK and DNAp occur in other forms in serum than during catalytic conditions in vitro. Finally, the guanosine analogs--acyclovir, bucyclovir, ganciclovir, (S)-9-(3,4-dihydroxybutyl)guanine, 9-(4-hydroxybutyl)guanine and (RS)-9-(4-hydroxy-2-(hydroxymethyl)butyl) guanine--were compared in enzyme kinetic experiments on varicella zoster virus (VZV) TK and TK2. They were also compared in their efficacy in inhibiting VZV multiplication in cell culture.
Keywords: Deoxyuracil Nucleotides/METABOLISM *DNA Replication Guanine/ANALOGS & DERIVATIVES Hela Cells Human Idoxuridine/*METABOLISM Kinetics Molecular Weight Substrate Specificity Thymidine Kinase/*METABOLISM *Virus Replication THESIS 900630
M9060641
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Important note: Information in this article was accurate in 1990. The state of the art may have changed since the publication date.
