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)]acety l-CoA (200 mCi/mmol). Using extracts of PC12 cells transiently transfe cted with a plasmid CMV-CAT, we found that the assay was linear with t ime for about 8 h, unless 25% of the substrate was exhausted. Under th e conditions of the assay, the tritiated substrate was relatively stab le, as 75% was still available for the reaction after a 20-h incubatio n at 37 degrees C under the toluene phase in the absence of cell extra ct. CAT activity could be reliably measured with 4-8 ng protein of cel l extract, corresponding to 50-100 transfected cells. We determined th e range of linearity of the initial rate with the volume of cell extra ct 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.