The expression mechanism of glutathione S-transferase in UV-B irradiated skin

UV-B酶、免疫印迹和免疫组织化学分析表明，脂肪皮肤胞浆中不含催化毒性产物η-6多不饱和脂肪酸残基脂质过氧化形成的同源、α类谷胱甘肽S转移酶A4-4的表达。经单次剂量(4000-24000mJ/cm2)紫外线(UVB，200mJ/cm2/min)照射后，大鼠皮肤中rGSTA4-4的表达水平为肝脏的五分之一，对HNE的表观比活性为24000ml/cm2。分离纯化rGSTA4-4皮肤，经反相分配高效液相色谱和CNBR裂解肽氨基酸序列分析鉴定其活性。多克隆抗体r-…免疫组织化学更多针对rGSTA4-4的研究表明，UVB照射后，rGSTA4-4在真皮中的表皮和皮脂腺中有选择性地表达。首次证明了ART酶BV-HNE的对映选择性失活和酶对HNE的对映选择性解毒。糖酵解酶3-磷酸甘油醛脱氢酶不可逆地被外消旋4-羟基-2(E)-壬烯醛(HNE)对映体选择性失活(S)，4-羟基-2(E)-壬烯醛(HNE)是细胞内脂质过氧化从生物膜释放的反应产物。(R)-HNE对GAPDH的IC50是S对映体的3.6倍。在大鼠肝细胞浆中，HNE被高度脱毒(S)-选择性地通过谷胱甘肽(GSH)结合和(R)-选择性地通过乙醇脱氢酶(ADH)介导的NADH依赖的还原。然而，在胞浆中，(R)-HNE的GSH结合比ADH介导的还原进行得更快。大鼠(R)肝脏中的次要谷胱甘肽S转移酶(GST)亚型A4-4在胞浆(S)选择性谷胱甘肽结合中起主要作用。纯化的rGSTA4-4对(S)-HNE的催化效率是(R)-HNE的4倍。由于(S)-HNE的Km比(R)-HNE小得多，以外消旋HNE为底物时，(S)-HNE优先被rGSTA4-4解毒。(S)-豚鼠和大鼠肝脏谷胱甘肽S-转移酶异构体优先解毒4-羟基-2(E)-壬烯醛(HNE)在豚鼠(GP)肝细胞质中，外消旋体4-羟基-2(E)-丁香烯醛(HNE)是生物膜通过脂质过氧化释放的一种活性高毒性产物，(S)优先受谷胱甘肽-S转移酶介导的谷胱甘肽结合和(R)-优先受乙醛脱氢酶介导的NAD+依赖的氧化作用。在豚鼠肝细胞浆中，GST介导的HNE对映体的解毒速率远高于ALDH。从豚鼠肝脏胞浆中分离到的主要gpGSTsA1-1、M1-1、M1-2和M1-3*也催化了HNE对映体的S优先偶联反应。豚鼠的肝脏和其他主要组织中没有GSTA4-4的免疫检测水平，GSTA4-4在大鼠(R)、小鼠和人的肝脏中作为一种少量的GST蛋白存在，对HNE表现出极高的催化活性。肝脏rGSTsA1-L(2)、A1-3、A4-4、M1-1、M1-2和M2-2也催化了HNE对映体的(S)优先结合反应。“更少

The expression of glutathione S-transferase A4-4 detoxifying 4-hvdroxy-2(E)-nonenal in the skin of rats irradiated by UV-BEnzyme, 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 … More 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 enzymesThe 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 ratsIn 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