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Glutathione and its Related Enzymes in the Nile Fish

Fish Physiology and Biochemistry volume 30, pages 189–199 (2004)Cite this article

Abstract

Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (K m) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC50 0.079, 3.98 and 0.126 μM, respectively.

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Abbreviations

CDNB:

1-chloro 2,4- dinitrobenzene

DEAE-cellulose:

diethylamino ethyl cellulose

EDTA:

ethylenediaminetetra acetic acid

GPx:

glutathione peroxidase

GR:

glutathione reductase

GSH:

reduced glutathione

GSSG:

disulfide glutathione

GST:

glutathione-S-transferase

IC50 :

inhibitor concentration causing 50% inhibition

NADPH:

nicotinamide adeninedinucleotide phosphate (reduced form)

SDS:

sodium dodecyl sulphate

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Authors and Affiliations

  1. Department of Molecular Biology, National Research Centre, Cairo, Egypt

    Ragaa R. Hamed & Rasha A. M. Guinidi

  2. Department of Biochemistry, Ain-Shams University, Egypt

    Tahany M. Maharem

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  1. Ragaa R. Hamed
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  2. Tahany M. Maharem
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  3. Rasha A. M. Guinidi
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Correspondence to Ragaa R. Hamed.

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Hamed, R.R., Maharem, T.M. & Guinidi, R.A.M. Glutathione and its Related Enzymes in the Nile Fish. Fish Physiol Biochem 30, 189–199 (2004). https://doi.org/10.1007/s10695-005-3259-5

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  • DOI: https://doi.org/10.1007/s10695-005-3259-5

Keywords

  • glutathione peroxidase
  • glutathione reductase
  • glutathione-S-transferase and purification

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