Abstract

N-Acetylation of xenobiotic-derived cysteineS-conjugates is a key step in the mercapturic acid pathway. The aim of this study was to investigate theN-acetylation of haloalkene-derivedS-haloalkyl and S-haloalkenyl cysteineS-conjugates by porcine kidney cysteineS-conjugate N-acetyltransferase (NAcT). A radioactive assay for the quantification of NAcT activity was developed as a new method for partial purification of the enzyme, which was necessitated by the substantial loss of activity during the immunoaffinity chromatography method. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate, rather thanN,N-bis[3-gluconamidopropyl]deoxycholamide, was used to solubilize the NAcT from porcine kidney microsomes in the revised procedure. The partially purified NAcT was free of detectable aminoacylase activity. Although low acetyl-coenzyme A hydrolase activity was observed, its effect on the assay was minimized by addition of excess acetyl-coenzyme A in the NAcT assay mixture. Attempts to separate the residual hydrolase activity from NAcT by different chromatographic procedures were either unsuccessful or lead to inactivation of NAcT. Most of the cysteineS-conjugates studied were N-acetylated by NAcT. Although the apparent K_m values for the cysteine S-conjugates studied differed by a factor of ∼2.5 (124–302 μM), a greater than 15-fold difference in the apparent V_max (0.75–15.6 nmol/h) andV_max/K_m(0.008–0.126 × 10⁻³ l h⁻¹) values was observed. These data show that a range of haloalkene-derived cysteineS-conjugates serve as substrates for pig kidney NAcT. The significant differences in cytotoxicity of these conjugates may be a result of more variable deacetylation rates of the corresponding mercapturates.