Original ArticlesHepatic mercapturic acid formation: involvement of cytosolic cysteinylglycine S-conjugate dipeptidase activity
Section snippets
Materials
mBCl and mBBr were purchased from Molecular Probes Europe BV (Leiden). DMSO was obtained from EGA-Chemie, N-acetylcysteine was from Fluka, and other chemicals and biochemicals from Merck, Sigma-Aldrich or Boehringer–Mannheim. SepPak-C18 cartridges were obtained from Millipore Corp.
Synthesis of bimane S-conjugates
The bimane S-conjugates of glutathione, cysteinylglycine, cysteine, and N-acetylcysteine were synthesized by reaction of mBBr with the respective thiols by a procedure modified from [17] as follows. Three milligrams
Mercapturic acid pathway enzyme activities measured with bimane S-conjugates in rat and guinea pig livers
Activities of the mercapturic acid pathway enzymes were measured in homogenates of rat and guinea pig liver (Table 1) using monochlorobimane and the respective bimane S-conjugates as substrates (Fig. 1). In rat liver, GST exhibited by far the highest activity (310 nmol × min−1 × mg of protein−1), demonstrating the high capacity of this organ for glutathione S-conjugate formation. Cysteinylglycine S-conjugate dipeptidase activity was also found to be relatively high (6.1 nmol × min−1 × mg of
Compartmentation of cysteinylglycine S-conjugate dipeptidase
Early studies on the subcellular distribution of dipeptide S-conjugate hydrolyzing activity performed on rat kidney demonstrated the existence of two different brush-border-membrane enzymes with activity toward cysteinylglycine S-conjugates, identified as aminopeptidase M and dehydropeptidase I 14, 15, 27. While the former was shown to be highly active in kidney, brain and small intestine, the latter showed high activity in lung and kidney [16]. In liver, both enzyme activities were only
Acknowledgements
The skillful technical assistance of Gisela Michael is gratefully acknowledged. The work was supported by a grant from the Deutsche Forschungsgemeinschaft (Grant Ak 8/2–1).
References (43)
- et al.
ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver
J Biol Chem
(1991) - et al.
Intrahepatic conversion of a glutathione conjugate to its mercapturic acidMetabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers
J Biol Chem
(1991) - et al.
Glutathione-degrading capacities of liver and kidney in different species
Biochem Pharmacol
(1990) - et al.
Specificity of a particulate rat renal peptidase and its localization along with other enzymes of mercapturic acid synthesis
Arch Biochem Biophys
(1978) - et al.
Glutathione-degrading enzymes of microvillus membranes
J Biol Chem
(1982) Tissue sulfhydryl groups
Arch Biochem Biophys
(1959)- et al.
Transport of glutathione disulfide and glutathione S-conjugates in hepatocyte plasma membrane vesicles
Meth Enzymol
(1994) - et al.
The relationship of biliary glutathione disulfide efflux and intracellular glutathione disulfide content in perfused rat liver
J Biol Chem
(1982) - et al.
Brush border membrane hydrolysis of S-benzylcysteine-p-nitroanilide, an activity of aminopeptidase M
Biochem Biophys Res Com
(1980) - et al.
γ-Glutamyl-p-nitroanilidea new convenient substrate for determination and study of L- and D-γ-glutamyltranspeptidase activities
Biochim Biophys Acta
(1963)