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MD Linetsky, F Hu, V Chemoganskiy, BJ Ortwerth; Studies on the Mechanism of the UVA Light-Dependent Loss of Glutathione Reductase Activity In Human Lenses . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3570.
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Purpose: To determine mechanism, which leads to UVA light-dependent loss of glutathione reductase activity (GR) in human lenses. Method: Intact human lenses (HL) in artificial aqueous humor, and the both HL water soluble (WS) fractions and yeast GR in 0.1 M KPO4 buffer, pH 7.0, were irradiated in a quartz cuvette with UVA light (200 mW/cm2/hr) for one hour at +20oC, and the specific activity (SA) was followed. GR apoenzyme was prepared by treatment of either HL WS fractions or yeast GR with (NH4)2SO4 (pH 2.25) in the cold. Reconstitution of apo-GR was conducted by mixing of enzyme with an excess of FAD, incubation and purification of GR on Sephadex G-25 column. Results: One hour of UVA photolysis of whole human lens resulted in a 75% decrease in the specific activity (SA) of GR (6.050.06 mU/mg lens protein vs 1.480.09 mU/mg lens protein). Similar drop in the SA was observed in the cortex (7412%) and in the nucleus (804%) of the lens after one hour of irradiation. Action spectra of GR SA in the WS fraction from HL within the range 320-500 nm showed that the enzyme was most vulnerable to the wavelengths in UVA region with the highest decrease in the SA at 320-350 nm (∼ 23-28% activity loss within 1 hour of irradiation), and lowest with the wavelengths beyond 400 nm (0-3% SA loss). UVA irradiation of GR apoenzyme in the crude HL WS fraction, followed by reconstitution with FAD, showed that 90% of the original SA could be recovered. The recovery of the activity of yeast GR, photolyzed under identical conditions, displayed essentially the same results. The original GR activity either from HL or from yeast GR, after UVA photolysis couldn't be recovered by (NH4)2SO4 (pH 2.25)-treatment when reconstituted with FAD. Experiments with UVA- photolyzed yeast GR have revealed that UVA photolysis caused the formation of the additional SH-groups within the enzyme, which was shown by an incorporation of highly SH-specific fluorescent probe, ABD-F, into the non- and UVA-irradiated GR under denaturing conditions. Conclusion: The results show that the loss of HL glutathione reductase SA mediated by UVA light is directly linked to the presence of FAD within the enzyme. The fact that the irradiated GR shows de novo formed SH-groups argues that UVA photolysis of glutathione reductase leads to the reduction of the redox active disulfide within the reaction center of the enzyme, making it inactive.
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