Modulation of firefly luciferase stability and impact on studies of gene regulation.
Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyl-transferase (CAT) and firefly luciferase (Luc). CAT has a half-life of about 50 h in mammalian cells, making it useful for transient transfection assays but less suitable for assays with stable cell lines. Luc has a half-life of only 3 h in mammalian cells, making it much more responsive in stable cell lines. Luc instability arises from its sensitivity to proteolysis both in vivo and in vitro. Compounds that resemble its natural substrate, luciferin, act as effective competitive inhibitors in vitro. When these compounds (e.g., phenylbenzothiazole) are added to either prokaryotic or eukaryotic cells, more than tenfold increases in Luc activity can be observed. This increased activity results from a lower rate of degradation of the enzyme in vivo and can be mimicked in vitro as phenylbenzothiazole protects Luc from trypsin digestion while it has no effect on the rate of digestion of alkaline phosphatase.
Thompson
J F
JF
Molecular Genetics and Protein Chemistry Department, Pfizer Inc., Groton, CT 06340.
Hayes
L S
LS
Lloyd
D B
DB
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D052160:Benzothiazoles; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006207:Half-Life; D006801:Humans; D007211:Indoles / Q000378:metabolism; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D052160:Benzothiazoles; D007211:Indoles; D010636:Phenols; D013844:Thiazoles; C070846:2-(2-hydroxyphenyl)benzothiazole; D049454:Firefly Luciferin; C057812:indoline; D008156:Luciferases; D000469:Alkaline Phosphatase; D014357:Trypsin; C070845:2-phenylbenzothiazole; C005465:benzothiazole