Studies on the mechanism of the UVA light-dependent loss of glutathione reductase activity in human lenses.
OBJECTIVE
To determine the mechanism that leads to the UVA light-dependent loss of glutathione reductase (GR) activity in human lens (HL).


METHODS
Both the HL water-soluble (WS) fraction and yeast GR were irradiated with UVA light (200 mW/(cm(2). h) for 1 hour at +20 degrees C, and the specific activity (SA) was observed. GR apoenzyme (apo-GR) was prepared from either HL-WS fractions or yeast GR by treatment with a cold solution of acidic ammonium sulfate. Reconstitution of apo-GR was conducted by mixing enzyme with an excess of flavine adenine dinucleotide (FAD) and purification of GR on a size-exclusion separation column.


RESULTS
One hour of UVA photolysis of an HL-WS fraction resulted in a 96% decrease in the SA of GR (6.32 +/- 0.22 vs. 0.39 +/- 0.01 mU/mg lens protein). Action spectra of GR SA in the WS fraction from HL within the range 320 to 500 nm showed that the enzyme was most vulnerable to the wavelengths in the UVA region with the highest decrease in the SA at 320 to 350 nm ( approximately 23%-28% activity loss within 1 hour of irradiation), and lowest with the wavelengths beyond 400 nm (7%-8% SA loss). UVA irradiation of apo-GR in the crude HL-WS fraction, followed by reconstitution with FAD, showed that 90% of the original SA was recovered. The original GR activity either in HL or yeast GR, however, was not recovered by (NH(4))(2)SO(4) (pH 2.25) treatment followed by reconstitution with FAD after UVA photolysis. Experiments with UVA-photolyzed yeast GR revealed that UVA photolysis caused the formation of additional SH groups within the enzyme, as shown by the incorporation of an SH-specific fluorescent probe, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Similar results were obtained on the photolyzed iodoacetamide-alkylated yeast GR, which was evaluated by matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry.


CONCLUSIONS
The results show that the reduction of HL GR activity by UVA light was directly linked to the presence of FAD within the enzyme. That the irradiated GR showed de novo formed SH groups argues that UVA photolysis of GR leads to the reduction of the redox-active disulfide within the reaction center of the enzyme, making it inactive.
Linetsky
Mikhail
M
Mason Eye Institute, University of Missouri, Columbia, Missouri 65201, USA. linetskym@health.missouri.edu
Hill
Jana M W
JM
Chemoganskiy
Vitaliy G
VG
Hu
Fang
F
Ortwerth
Beryl J
BJ
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000201:enzymology* / Q000528:radiation effects*; D010069:Oxadiazoles; D010782:Photolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014466:Ultraviolet Rays
D010069:Oxadiazoles; D005182:Flavin-Adenine Dinucleotide; C046364:4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; D005980:Glutathione Reductase