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Characterization of a Novel Glutathione S-Transferase, Which May Protect From Oxidative Stress
EPA Grant Number: U915006Title: Characterization of a Novel Glutathione S-Transferase, Which May Protect From Oxidative Stress
Investigators: Piper, John T.
Institution: University of Texas Medical Branch - Galveston
EPA Project Officer: McClure, Karen
Project Period: January 1, 1996 through December 7, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996)
Research Category: Fellowship - Toxicology , Health Effects , Academic Fellowships
Description:
Objective:The objective of this research project is to investigate and structurally and kinetically characterize the physiological relevance of a novel glutathione S-transferase (GST) isozyme, which may play a significant role in the protection from both chemically induced oxidative stress and the deleterious end products of this process.
Approach:The tissue distribution of hGST 5.8 will be assessed by standard immunodetection techniques. The primary structure of hGST 5.8 will be determined in parallel by both protein chemistry and molecular biology techniques. The liver and aorta forms of hGST 5.8 will be purified to homogeneity, and the primary sequence of the purified protein will be determined through a series of digests with subsequent sequencing of the resultant peptide fragments. The cDNA of liver hGST 5.8 will be obtained through screening of a commercially prepared cDNA library using the cDNA of the mouse homolog of hGST 5.8, mGSTA4-4, as a probe. The recombinant hGST 5.8 protein then will be expressed in a prokaryotic (nonnucleated) expression system. Once the kinetic characteristics of the recombinant protein have been established and compared to the native enzyme, extensive kinetic studies using various GST substrates and inhibitors will be performed.
The physiological relevance of hGST 5.8 as a protective mechanism against oxidative stress will be investigated using stable transection of a eukaryotic (nucleated) hGST 5.8 expression construct into H-69 cells. Once stable subclonal cell lines have been established, the cells will be exposed to increasing levels of a variety of compounds known to cause oxidative stress. Cell survival, glutathione levels, and lipid peroxidation products in the form of thiobarbituric acid reactive species will be compared in control and treated cells to determine if the over expression of hGST 5.8 can provide increased protection from oxidative stress.
Supplemental Keywords:fellowship, glutathione-s-transferase, GST, oxidative stress, acid reactive species.