Selective inactivation of eukaryotic beta-galactosidase in assays for inhibitors of HIV-1 TAT using bacterial beta-galactosidase as a reporter enzyme.
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.
Young
D C
DC
Department of Virology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426.
Kingsley
S D
SD
Ryan
K A
KA
Dutko
F J
FJ
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002021:Buffers; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000032:analysis; D015696:Gene Products, tat / Q000037:antagonists & inhibitors*; D005798:Genes, Bacterial / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012474:Salmon; D012680:Sensitivity and Specificity; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001426:Bacterial Proteins; D002021:Buffers; D004791:Enzyme Inhibitors; D015696:Gene Products, tat; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D008274:Magnesium; D011188:Potassium