A method for increasing the sensitivity of chloramphenicol acetyltransferase assays in extracts of transfected cultured cells.
Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60 degrees C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002457:Cell Extracts / Q000378:metabolism; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D006358:Hot Temperature; D006801:Humans; D006868:Hydrolysis; D014162:Transfection
D002457:Cell Extracts; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase