| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
The expression of glutathione S-transferase A4-4 detoxifying 4-hvdroxy-2(E)-nonenal in the skin of rats irradiated by UV-B
Enzyme, Western blot, and immunohistochemical analyzes indicated that fat skin cytosol contained no detectable level of the homodimefic, Alpha-class glutathione S-transferase(rGST) A4-4 which catalyzes the GSH conjugation of the toxic product, 4-hydroxy-2(E)-nonenal(HNE), non-enzymatically formed from η-6 polyunsaturated fatty acid residues of lipids by lipid peroxidation. Rats irradiated by single doses(4, 000-24, 000 mJ/cm2) of ultraviolet B-band light(UVB, 200 mJ/cm2/min) markedly expressed rGSTA4-4 in the skin at a level of one fifth of that of the liver in apparent specific activity toward HNE at a single dose of 24, 000 ml/cm2.Skin rGSTA4-4 was isolated, purified to homogeneity, and identified with hepatic rGSTA4-4 by reverse phase partition HPLC and by amino acid sequence analysis of its CNBr fission peptides. Imiriunohistochemistry with polyclonal antibody r
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aised against rGSTA4-4 demonstrated the selective expression of rGSTA4-4 in epidermis and sebaceous glands localized in dermis after UVB irradiation.
The first evidence for the enantioselective mactivation of art enzyme bv HNE and for the enantioselective detoxification of HNE by enzymes
The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase(GAPDH), was irreversibly and highly(S)-selectively inactivated by the enantiomers of racemic 4-hydroxy-2(E)-nonenal(HNE), a reactive product released from biomembranes by lipid peroxidation in cells. IC50 of(R)-HNE for GAPDH was 3.6-fold higher than that of the(S)-enantiomer. In rat liver cytosol, the HNE was detoxified highly(S)-selectively by glutathione(GSH) conjugation and(R)-selectively by NADH-dependent reduction mediated by alcohol dehydrogenase(ADH). In the cytosol, however, the GSH conjugation of(R)-HNE proceeded at a much higher rate than did its ADH-mediated reduction. The minor glutathione S-transferase(GST)isoform, A4-4, in the rat(r)liver played a major role in cytosolic (S)-selective GSH conjugation. The catalytic efficiency, kcat/Km, of purified rGSTA4-4 was 4-fold higher for (S)-HNE than for (R)-HNE. Because of its much smaller Km than that of (R)-HNE, (S)-HNE was preferentially detoxified by rGSTA4-4 when racemic HNE was used as a substrate.
(S)-Preferential detoxification of 4-hvdroxv-2(E)-nonenal enantiomers by hepatic glutathione S-transferase isoforms in guinea pigs and rats
In guinea pig(gp) liver cytosol, racemic 4-hydroxy-2(E)-ilonenal(HNE), a reactive and highly toxic product released from biomembranes by lipid peroxidation, was detoxified(S)preferentially by glutathione(GSH) conjugation mediated by GSH S-transferases(GSTs) and(R)-preferentially by NAD+-dependent oxidation mediated by aldehyde dehydrogenase (ALDH). The GST-mediated detoxification of the HNE enantiomers proceeded at much higher rates than that mediated by ALDH in guinea pig liver cytosol. All the major gpGSTsA1-1, M1-1, M1-2, and M1-3* isolated from guinea pig liver cytosol also catalysed the (S)-preferential conjugation of the HNE enantiomers. The liver and other major tissues of guinea pigs had no immunologically detectable level of a GSTA4-4 ortholog which exists as a minor GST protein in the rat(r), mouse, and human liver and exhibits extremely high catalytic activity towards HNE. All the hepatic rGSTsA1-l(2), A1-3, A4-4, M1-1, M1-2, and M2-2 also catalysed the (S)-preferential conjugation of HNE enantiomers. " Less
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