Elsevier

Biochemical Pharmacology

Volume 56, Issue 6, 15 September 1998, Pages 763-771
Biochemical Pharmacology

Original Articles
Hepatic mercapturic acid formation: involvement of cytosolic cysteinylglycine S-conjugate dipeptidase activity

https://doi.org/10.1016/S0006-2952(98)00065-3Get rights and content

Abstract

The role of cysteinylglycine S-conjugate dipeptidases in the intrahepatic mercapturic acid pathway was investigated in rat liver. Subcellular compartmentation studies and liver perfusions were performed using monochlorobimane and bimane S-conjugates as model compounds. The major part (over 95%) of total hepatic cysteinylglycine S-conjugate dipeptidase activity was located in the cytosol. Lower specific activity appeared in the canalicular plasma membrane fraction. Similar hepatic localization of dipeptidase activity was seen in the guinea pig. In intact rat liver perfused with monochlorobimane, the major products were the glutathione S-conjugate (mBSG) and the cysteinylglycine S-conjugate (mBCG) in bile. Minor amounts of the cysteine S-conjugate (mBCys) and the mercapturic acid (mBNAc) were formed, indicating a limitation in further metabolism of the dipeptide S-conjugate in the biliary space. However, when the dipeptide S-conjugate was offered to the sinusoidal space in liver perfusions, substantial uptake and conversion to mBNAc was observed, and only trace amounts of the infused dipeptide appeared in bile. The data suggest that cytosolic cysteinylglycine S-conjugate dipeptidase as identified here is involved in hepatic mercapturic acid formation from sinusoidal cysteinylglycine S-conjugates. This is especially of significance for species such as guinea pig and human, in which dipeptide S-conjugates are generated in the sinusoidal domain of the liver due to the presence of high γ-glutamyltranspeptidase 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)

  • W.H Habig et al.

    Glutathione S-transferases

    J Biol Chem

    (1974)
  • A Bensadoun et al.

    Assay of proteins in the presence of interfering materials

    Anal Biochem

    (1976)
  • M.E Horbach et al.

    Identification of a hepatic plasma membrane glutathione S-transferase activated by N-ethylmaleimide

    Biochim Biophys Acta

    (1993)
  • H Adachi et al.

    Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells

    Biochim Biophys Acta

    (1992)
  • G.M Habib et al.

    Four distinct membrane-bound dipeptidase RNAs are differentially expressed and show discordant regulation with γ-glutamyltranspeptidase

    J Biol Chem

    (1996)
  • M Büchler et al.

    cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats

    J Biol Chem

    (1996)
  • J.L Stevens et al.

    The mercapturic acid pathwaybiosynthesis, intermediary metabolism and physiological disposition

  • W Dekant et al.

    Formation and fate of nephrotoxic and cytotoxic glutathione S-conjugatesCysteine conjugate β-lyase pathway

    Adv Pharmacol

    (1994)
  • C.A Hinchman et al.

    Glutathione conjugation and conversion to mercapturic acids can occur as an intrahepatic process

    J Toxicol Environ Health

    (1994)
  • J.N.M Commandeur et al.

    Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates

    Pharmacol Rev

    (1995)
  • A Wahlländer et al.

    Glutathione S-conjugate formation from 1-chloro-2,4-dinitrobenzene and biliary excretion in the perfused rat liver

    Eur J Biochem

    (1979)
  • Cited by (0)

    View full text