Vol. 28, Issue 4, 440-445, April 2000

Porcine Kidney Microsomal Cysteine S-Conjugate N-Acetyltransferase-Catalyzed N-Acetylation of Haloalkene-Derived Cysteine S-Conjugates

Torsten Kraus, Viuita Uttamsingh, M.W. Anders, and Sabine Wolf

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York (V.U., M.W.A.); and Institut für Biochemie, Technische Universität Darmstadt, Darmstadt, Germany (T.K., S.W.)

N-Acetylation of xenobiotic-derived cysteine S-conjugates is a key step in the mercapturic acid pathway. The aim of this study was to investigate the N-acetylation of haloalkene-derived S-haloalkyl and S-haloalkenyl cysteine S-conjugates by porcine kidney cysteine S-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 than N,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 cysteine S-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) and V_max/K_m (0.008-0.126 × 10³ l h¹) values was observed. These data show that a range of haloalkene-derived cysteine S-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.