Assaying the reporter gene chloramphenicol acetyltransferase

David W. Crabb, Carolyn D. Minth, Jack E. Dixon

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved.

Original languageEnglish (US)
Pages (from-to)690-701
Number of pages12
JournalMethods in Enzymology
Volume168
Issue numberC
DOIs
StatePublished - Jan 1 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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