Document 0819
 DOCN  M9440819
 TI    Selective inactivation of eukaryotic beta-galactosidase in assays for
       inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter
       enzyme.
 DT    9404
 AU    Young DC; Kingsley SD; Ryan KA; Dutko FJ; Department of Virology,
       Sterling Winthrop Pharmaceuticals; Research Division, Collegeville,
       Pennsylvania 19426.
 SO    Anal Biochem. 1993 Nov 15;215(1):24-30. Unique Identifier : AIDSLINE
       MED/94127699
 AB    Bacterial beta-galactosidase is one of several reporter enzymes used in
       studying the transcriptional activity of eukaryotic promoters. Although
       it is one of the easiest and least expensive enzymes to assay, its use
       has been limited because of its low sensitivity, which is due in part to
       endogenous levels of beta-galactosidase in many eukaryotic cells. In
       this study, we compared the pH and salt requirements, as well as the
       heat stability, of bacterial and eukaryotic beta-galactosidase in order
       to identify conditions which would inhibit the beta-galactosidase enzyme
       endogenous to eukaryotic cells without adversely affecting the activity
       of either purified bacterial beta-galactosidase or reporter
       beta-galactosidase produced after transfection of expression vectors
       into eukaryotic cells. Heat treatment at 50 degrees C for 1 h
       inactivated the beta-galactosidase activity endogenous to several
       eukaryotic cell lines by as much as 40-fold without adversely affecting
       the activity of bacterial beta-galactosidase. This treatment increased
       the sensitivity of this reporter enzyme and allowed the development of a
       rapid and quantifiable screening assay for HIV-1 tat inhibitors.
 DE    beta-Galactosidase/ANTAGONISTS & INHIB/*METABOLISM  Animal  Bacterial
       Proteins/GENETICS/METABOLISM  Buffers  Chloramphenicol
       Acetyltransferase/GENETICS/METABOLISM  Comparative Study  Enzyme
       Activation  Enzyme Inhibitors/ANALYSIS  Gene Products, tat/*ANTAGONISTS
       & INHIB  Genes, Bacterial/GENETICS  Hela Cells  Human  Hydrogen-Ion
       Concentration  Magnesium/PHARMACOLOGY  Potassium/PHARMACOLOGY  Salmon
       Sensitivity and Specificity  Temperature  Transfection  Tumor Cells,
       Cultured  Vero Cells  JOURNAL ARTICLE

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