Porcine kidney microsomal cysteine S-conjugate N-acetyltransferase-catalyzed N-acetylation of haloalkene-derived cysteine S-conjugates

• Published in: Drug metabolism and disposition Vol. 28; no. 4; pp. 440 - 445
• Main Authors: KRAUS, T, UTTAMSINGH, V, ANDERS, M. W, WOLF, S
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.04.2000
• Subjects: Acetyl-CoA Hydrolase - metabolism; Acetylation; Acetyltransferases - metabolism; Alkenes - metabolism; Amidohydrolases - metabolism; Animals; Biological and medical sciences; Chemical and industrial products toxicology. Toxic occupational diseases; Cysteine - metabolism; General pharmacology; In Vitro Techniques; Kidney - enzymology; Kinetics; Medical sciences; Microsomes - enzymology; Miscellaneous; Pharmacology. Drug treatments; Swine; Toxicology; Various organic compounds; Urine; Acyltransferases; Cysteine-S-conjugate N-acetyltransferase; Biological fluid; Enzyme; Transferases; Elimination; Mercaptoacid; Microsome; Metabolism; In vitro; Kidney; Pig; Toxicokinetics; Vertebrata; Mammalia; Urinary system; Animal; Artiodactyla; Acetylation; Ungulata; Xenobiotic
• ISSN: 0090-9556; 1521-009X

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 approximately 2.5 (124-302 microM), 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 x 10(-3) l h(-1)) 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.