Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

A simplified radioenzymatic assay for chloramphenicol was developed by eliminating the need for cumbersome extraction procedures. After the acetylation of chloramphenicol with [(14)C]acetyl coenzyme A in the presence of chloramphenicol acetyltransferase, the reaction mixture was added to a toluene-based scintillation fluid. Since (14)C-acetylated chloramphenicol is more soluble than [(14)C]acetyl coenzyme A in toluene, the radioactive product could be counted directly. The rapidity of this assay, as well as its accuracy, precision, and specificity, makes it particularly suitable for clinical use. In contrast to previous reports of enzymatic assays for chloramphenicol, we have found that results of the assay of standards prepared in serum were up to 25% higher than those of standards prepared in saline, cerebrospinal fluid, or urine.
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PMID:Simplified radioenzymatic assay for chloramphenicol. 62 88

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail. 159 93

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.
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PMID:A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells. 275 94

We have explored the possibility of increasing the sensitivity of the mixed-phase assay for chloramphenicol acetyltransferase (CAT) by using a low concentration (3.75 microM) of isotopically undiluted [3H]acetyl-CoA (200 mCi/mmol). Using extracts of PC12 cells transiently transfected with a plasmid CMV-CAT, we found that the assay was linear with time for about 8 h, unless 25% of the substrate was exhausted. Under the conditions of the assay, the tritiated substrate was relatively stable, as 75% was still available for the reaction after a 20-h incubation at 37 degrees C under the toluene phase in the absence of cell extract. CAT activity could be reliably measured with 4-8 ng protein of cell extract, corresponding to 50-100 transfected cells. We determined the range of linearity of the initial rate with the volume of cell extract and showed that, above a certain value, the rate becomes limited by the diffusion of 3H-acetylated chloramphenicol in the organic phase. The sensitivity of the new assay compared favorably with that of the previously described CAT assays and approached that of the luciferase assay.
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PMID:Performance and limits of the mixed-phase assay for chloramphenicol acetyltransferase at low [3H]acetyl-CoA concentration. 805 42