| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
Molecular cloning of a cDNA and characterization of a recombinant rat theta-class glutathione S-transferase Yrs-Yrs
A cDNA containing the entire coding sequence for the subunit protein of rat liver class theta glutathione S-transferase (GST) Yrs-Yrs was isolated from a rat liver lambdagt 11 cDNA library. The cDNA,designated GST thetal, consisted of 1,258 bp which had an open reading frame of 732 bp encoding a polypeptide of 244 amino acid (AA) residues, including the leading AA Met to be removed on expression. The authenticity of the cDNA structure was supported by matching its deduced AA sequence with N-termini of Yrs and peptides obtained thereof by tryptic digestion as well as by CNBr cleavage. The deduced AA sequence of the subunit Yrs (M.W.27,311) had only a weak homology (19-23%) with those of rat liver classes alpha, mu, and pi GST isozymes. The theta-1 cDNA was expressed in Escherichia coli. The recombinant isozyme was purified to apparent homogeneity by the method previously re
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ported for the isolation of Yrs-Yrs from rat liver cytosol. The recombinant enzyme had significant activity toward cumene hydroperoxide, arachidonic acid 15-hydroperoxide, 1-menaphthyl sulfate, and SMCR.
Tissue distribution of GST Yrs-Yrs in rats, mice, hamsters and guinea pigs
GST activity toward SMCR was simultaneously examined in various organ cytosol fractions from rats, mice hamsters or guinea pigs. Liver cytosols from those animals had the highest activity toward SMCR.There was a marked difference in the order of activity in those cytosol fractions using SMCR as substrate (rat>mouse>>hamster=guinea pig). However, there was little sex defferences in the hepatic GST activity towar SMCR in four animal species when the enzyme activity was assayd under the same conditions of substrate and GSH concentration. Western blot analyzes of the cytosols from various tissues of the above animal species indicated that the tissue level of GST Yrs-Yrs was as follows : liver>testis>adrenal>kidney>lung>brain>skeletal muscle=heart=small intestine=spleen=skin=0. No protein cross-reacting with anti-GST Yrs-antisera was determined with SMCR were in good accordance with the result of the Western blot analysis.
Simultaneous isolation and purification of theta-class GSTs 5-5 and Yrs-Yrs from rat liver cytosol
A method was established for simultaneously isolating theta-class GSTs (GST 5-5 and Yrs-Yrs) as homogeneous proteins from rat liver cytosol. The established method using an 8-aminooctyl Sepharose 4B column to separate 5-5 from Yrs-Yrs at the final stage of their purification was a modification of the method previously reported for the isolation of Yrs-Yrs. Specific substrates used for purification of the theta-class GSTs were dichloromethane for 5-5 and SMCR for Yrs-Yrs. GSTs 5-5 and Yrs-Yrs existed at a ratio of 1 : 7 at a total concentration of 0.5% of that of the cytosolic protein. Western blot analysis indicated that rabbit antisera raised against GSTs 5-5 and Yrs-Yrs intensely cross-reacted with the corresponding antigens, but showed no detectable cross-reactivity with the different subfamilies of theta-class GSTs as well as with representative rat liver GSTs of other classes. The theta-class GSTs showed higher GSH peroxidase activity than rat GST Ya-Ya of the alpha-class toward hydroperoxides of cumene, arachidonic acid, and linoleic acid. Cumene hydroperoxide was a better substrate for 5-5 than for Yrs-Yrs, while the fatty acid hydroperoxides were the better substrates for Yrs-Yrs than for 5-5. Less
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