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Human lens thioltransferase: cloning, purification, and function.
Invest Ophthalmol Vis Sci. 2001 Mar; 42(3):743-51.IO

Abstract

PURPOSE

To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT).

METHODS

The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2.

RESULTS

The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells.

CONCLUSIONS

The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress.

Authors+Show Affiliations

Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, 134 VBS, Lincoln, NE 68583-0905, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11222536

Citation

Qiao, F, et al. "Human Lens Thioltransferase: Cloning, Purification, and Function." Investigative Ophthalmology & Visual Science, vol. 42, no. 3, 2001, pp. 743-51.
Qiao F, Xing K, Liu A, et al. Human lens thioltransferase: cloning, purification, and function. Invest Ophthalmol Vis Sci. 2001;42(3):743-51.
Qiao, F., Xing, K., Liu, A., Ehlers, N., Raghavachari, N., & Lou, M. F. (2001). Human lens thioltransferase: cloning, purification, and function. Investigative Ophthalmology & Visual Science, 42(3), 743-51.
Qiao F, et al. Human Lens Thioltransferase: Cloning, Purification, and Function. Invest Ophthalmol Vis Sci. 2001;42(3):743-51. PubMed PMID: 11222536.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Human lens thioltransferase: cloning, purification, and function. AU - Qiao,F, AU - Xing,K, AU - Liu,A, AU - Ehlers,N, AU - Raghavachari,N, AU - Lou,M F, PY - 2001/2/27/pubmed PY - 2001/3/17/medline PY - 2001/2/27/entrez SP - 743 EP - 51 JF - Investigative ophthalmology & visual science JO - Invest Ophthalmol Vis Sci VL - 42 IS - 3 N2 - PURPOSE: To clone the human lens thioltransferase (TTase) gene and to purify, characterize and study the possible function of the recombinant human lens thioltransferase (RHLT). METHODS: The human lens TTase gene was cloned by using RT-PCR and verified by sequence and RNase protection assay. TTase overexpressed in Escherichia coli was isolated and purified to homogeneity by column chromatography and identified by Western blot analysis. The activity was assayed with a synthetic substrate hydroxyethyl disulfide. Its function in dethiolating and reactivating other key metabolic enzymes was studied by using pure glutathione S:-transferase (GST) and glutathione peroxidase (GPx) from commercial source and also with the cell extract of rabbit lens epithelial cells preexposed to H2O2. RESULTS: The cloned human lens TTase gene showed identical sequence to the TTase gene from other human tissues. The RNase protection assay displayed a single transcript from the total RNA of human lens epithelial cells. The purified RHLT had a molecular weight of 11.8 kDa and reacted positively with anti-pig liver TTase. It displayed similar structural, functional, and kinetic characteristics to those of TTases from other sources. It was shown that RHLT effectively regenerated the activities of GST and GPx, after each was inactivated by S-thiolation with cystine in vitro. Furthermore, RHLT was able to restore the activity of the oxidatively inactivated glyceraldehyde-3-phosphate dehydrogenase (G-3PD) in H2O2-exposed rabbit lens epithelial cells. CONCLUSIONS: The human lens TTase gene has been cloned for the first time. Its gene product showed the characteristics which support our speculation that TTase may play a major role in maintaining the homeostasis of lens protein thiols thus protecting against oxidative stress. SN - 0146-0404 UR - https://www.unboundmedicine.com/medline/citation/11222536/Human_lens_thioltransferase:_cloning_purification_and_function_ L2 - https://iovs.arvojournals.org/article.aspx?volume=42&issue=3&page=743 DB - PRIME DP - Unbound Medicine ER -