MODULATION OF FIREFLY LUCIFERASE STABILITY AND IMPACT ON STUDIES OF GENE-REGULATION
MODULATION OF FIREFLY LUCIFERASE STABILITY AND IMPACT ON STUDIES OF GENE-REGULATION
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DOI：
10.1016/0378-1119(91)90270-l
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发表时间：
1991-07-22
期刊：
影响因子：
3.5
通讯作者：
LLOYD, DB
中科院分区：
文献类型：
--
作者：
THOMPSON, JF;HAYES, LS;LLOYD, DB
在真核基因表达研究中最常用的两个记者酶是氯霉素乙酰基转移酶（CAT）和萤火虫荧光素酶（LUC）。 CAT在哺乳动物细胞中的半衰期约为50小时，使其可用于瞬时转染测定法，但不适合具有稳定细胞系的测定法。 LUC在哺乳动物细胞中只有3小时的半衰期，使其在稳定的细胞系中的响应性更高。 Luc不稳定性源于体内和体外对蛋白水解的敏感性。 类似于天然底物的化合物在体外充当有效的竞争抑制剂。 当这些化合物（例如，将苯基苯甲酰唑）添加到原核细胞或真核细胞中时，可以观察到LUC活性的十倍以上。 这种增加的活性是由于体内酶的降解率较低而导致的，并且可以在体外模仿，因为苯基苯甲唑可保护LUC免受胰蛋白酶消化的影响，而对碱性磷酸酶消化的速度无效。
Two of the reporter enzymes most commonly used in studies of eukaryotic gene expression are chloramphenicol acetyltransferase (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.