Novel human ocular glutathione S-transferases with high activity toward 4- hydroxynonenal

S. S. Singhal, S. Awasthi, S. K. Srivastava, P. Zimniak, Naseem Ansari, Y. C. Awasthi

Research output: Contribution to journalArticle

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Abstract

Purpose. To study the distribution and expression of glutathione S- transferase isozymes involved m detoxification of endogenously generated toxic products of lipid peroxidation, namely, 4-hydroxynonenal (4-HNE) in human lens, retina, cornea, iris, ciliary body and to study their kinetic and structural properties. Methods. The authors have previously cloned and sequenced cDNA of mouse mGSTA4-4, which shows high activity towards 4-HNE. They have expressed it in Escherichia coli and have raised antibodies against the recombinant mGSTA4-4. In the present study, these antibodies were used in Western blot analysis and immunoaffinity chromatography to study the expression and to purify the human ortholog(s) of mGSTA4-4 from ocular tissues. Results. Western blot analyses of human ocular tissues indicated that a glutathione S-transferases (GST) isozyme immunologically similar to mGSTA4-4 was expressed in cornea, retina, and iris and ciliary body, but not in lens. This isozyme designated as hGST 5.8 was purified to homogeneity from human retina, cornea, and iris and ciliary body by immunoabsorption on immobilized antibodies against mGSTA4-4. The human ortholog of mGSTA4-4, designated as hGST 5.8 purified from all these tissues and pI value of 5.8, subunit Mr value of 25 k and blocked N-terminal. Amino acid sequences of CNBr fragments of hGST 5.8 isozymes of human ocular tissues showed a high degree of primary structure homologies with the corresponding regions of mGSTA4-4. There were noticeable differences in the amino acid sequences of hGST 5.8 of cornea, retina, and iris and ciliary body, suggesting the presence of several closely related hGST 5.8 subunits in the ocular tissues. This heterogeneity was due to tissue-specific expression rather than simple allelic polymorphism. The hGST 5.8 had about sixfold to eightfold higher activity toward 4-hydroxynonenal than I-chloro-2,4-dinitrobenzene, or CDNB. The catalytic efficiency (Kcat/Km) of ocular hGST 5.8 for 4-HNE was about 100- fold higher than those for the α, μ, or π classes of GST. In addition, hGST 5.8 expressed glutathione peroxidase activity toward phospholipid hydroperoxides and GSH-conjugating activity toward 9,10-epoxy stearic acid. Conclusions. The results indicate that hGST 5.8 isozyme(s) distinct from the α, μ , and π classes of GSTs, are differentially expressed in human ocular tissues and stay play an important role in protective mechanisms against endogenous toxicants generated during lipid peroxidation.

Original languageEnglish (US)
Pages (from-to)142-150
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume36
Issue number1
StatePublished - 1995

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Glutathione Transferase
Ciliary Body
Isoenzymes
Iris
Cornea
Retina
Lipid Peroxidation
Lenses
Amino Acid Sequence
Western Blotting
Immobilized Antibodies
Antibodies
Poisons
Glutathione Peroxidase
mGSTA4-4
4-hydroxy-2-nonenal
Hydrogen Peroxide
Chromatography
Phospholipids
Complementary DNA

Keywords

  • 4-hydroxynonenal
  • glutathione S-transferases
  • human ocular tissues
  • lipid peroxidation

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Singhal, S. S., Awasthi, S., Srivastava, S. K., Zimniak, P., Ansari, N., & Awasthi, Y. C. (1995). Novel human ocular glutathione S-transferases with high activity toward 4- hydroxynonenal. Investigative Ophthalmology and Visual Science, 36(1), 142-150.

Novel human ocular glutathione S-transferases with high activity toward 4- hydroxynonenal. / Singhal, S. S.; Awasthi, S.; Srivastava, S. K.; Zimniak, P.; Ansari, Naseem; Awasthi, Y. C.

In: Investigative Ophthalmology and Visual Science, Vol. 36, No. 1, 1995, p. 142-150.

Research output: Contribution to journalArticle

Singhal, SS, Awasthi, S, Srivastava, SK, Zimniak, P, Ansari, N & Awasthi, YC 1995, 'Novel human ocular glutathione S-transferases with high activity toward 4- hydroxynonenal', Investigative Ophthalmology and Visual Science, vol. 36, no. 1, pp. 142-150.
Singhal, S. S. ; Awasthi, S. ; Srivastava, S. K. ; Zimniak, P. ; Ansari, Naseem ; Awasthi, Y. C. / Novel human ocular glutathione S-transferases with high activity toward 4- hydroxynonenal. In: Investigative Ophthalmology and Visual Science. 1995 ; Vol. 36, No. 1. pp. 142-150.
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AU - Awasthi, S.

AU - Srivastava, S. K.

AU - Zimniak, P.

AU - Ansari, Naseem

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