PERFORMANCE AND LIMITS OF THE MIXED-PHASE ASSAY FOR CHLORAMPHENICOL ACETYLTRANSFERASE AT LOW [H-3] ACETYL-COA CONCENTRATION

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 mu M) of isotopically undiluted [3(H)]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 H-3-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. (C) 1994 Academic Press, Inc.