Porcine kidney microsomal cysteine S-conjugate N-acetyltransferase-catalyzed N-acetylation of haloalkene-derived cysteine S-conjugates.
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.
Kraus
T
T
Institut für Biochemie, Technische Universität Darmstadt, Darmstadt, Germany.
Uttamsingh
V
V
Anders
M W
MW
Wolf
S
S
D000104:Acetyl-CoA Hydrolase / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000475:Alkenes / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D013552:Swine
D000475:Alkenes; D000123:Acetyltransferases; C034954:cysteine S-conjugate N-acetyltransferase; D000104:Acetyl-CoA Hydrolase; D000581:Amidohydrolases; C019672:aminoacylase I; D003545:Cysteine