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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies of the transcriptional activity of gene promoters have been greatly assisted by the widespread use of the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. Previous techniques for assaying CAT enzymatic activity have utilized radioactive substrates or cofactors with the resulting complications of handling radioactive materials. We report here the development of fluorescent substrates for the CAT enzyme which form the basis of a CAT enzyme assay of enzyme kinetic parameters (Km and Vmax) and sensitivity similar to those based on radioactive substrates. Fluorescent substrates were designed as analogs of chloramphenicol and were based on the structure-function requirements of the enzyme. Several fluorophores were used to derivatize chloramphenicol base; one of the most effective was the borondipyrromethene difluoride (BODIPY) fluorophore. One BODIPY-chloramphenicol analog was found to have a Km for the purified CAT enzyme of 2 microM (compared to 12 microM for 14C-labeled chloramphenicol) and a Vmax of 120 pmole/min (compared to 180 pmol/min for the radioactive substrate). To verify its usefulness, a BODIPY--chloramphenicol-based CAT assay was used to measure transient transfection of primary cultures of ovarian granulosa cells in serum-free medium. This experimental system requires a highly sensitive assay for detecting transfected CAT gene activity. Robust expression of CAT activity was easily detected in crude cellular extracts using FluoReporter FAST CAT, a kit containing the BODIPY-chloramphenicol analog. The expression was precisely quantified by methanol extraction of the substrate and products from TLC plates and subsequent measurement of fluorescence using excitation-emission spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A nonradioactive assay for transfected chloramphenicol acetyltransferase activity using fluorescent substrates. 178 95

This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA (or any of several other acyl CoA cofactors) to chloramphenicol. In the assays described here, transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material using either thin-layer chromatography or phase-extraction. The second reporter system uses a kit to perform a simple two-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.
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PMID:Isotopic assays for reporter gene activity. 1826 85

This unit describes two widely used reporter systems that are based on radioactive detection assays. The first assay uses chloramphenicol acetyltransferase (CAT) activity as a measure of the level of expression of a transfected gene. This bacterial enzyme catalyzes the transfer of an acyl group from acetyl CoA to chloramphenicol. Transfected cells are harvested and lysed, and then acyl CoA and radioactively labeled chloramphenicol are added to cell lysate, and modified derivatives of the antibiotic are separated from the starting material. The second reporter system uses a kit to perform a simple two-site radioimmunoassay to quantitate the amount of human growth hormone (hGH) secreted into culture medium by transfected cells. Medium is incubated with 125I-labeled antibody specific for hGH, and immune complexes are collected by an avidin-coated bead. The quantity of hormone is determined based on comparison with a standard curve.
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PMID:Isotopic assays for reporter gene activity. 2196 75