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TB Estey, AE Pappa, JF Carpenter, VK Vasiliou; Biochemical and Biophysical Characterization of UV-Exposed Human ALDH3A1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1645.
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PURPOSE: Mammalian corneal epithelium expresses in abundance a cytosolic aldehyde dehydrogenase (ALDH3A1), which apparently protects the eye from the UV-induced tissue damage. In mice, it has been shown that UV-light decreases corneal ALDH3A1 enzymatic activity, and this decrease is associated with corneal clouding. The aim of this study was to elucidate the effects of UV-exposure on the structure and function of the human ALDH3A1 in vitro. METHODS: A recombinant baculovirus was constructed containing human ALDH3A1 cDNA and used to produce a functional enzyme in Spodoptera frugiperda (Sf 9) cells. Purification of ALDH3A1 was accomplished using affinity chromatography and yielded a single band on SDS-PAGE gels at 54 kDa. The purified protein was irradiated at 295 nm, a wavelength characteristic of UVB-light, for varying time intervals. Structural characterization of UV-exposed ALDH3A1 included limited proteolysis, far-UV circular dichroism, intrinsic fluorescence, SEC-HPLC, and FTIR analysis. RESULTS: UV-exposure dramatically reduced the enzymatic activity of ALDH3A1. The extent of this reduction is proportional to UV-exposure time and characterized by a decrease in Vmax, though both kcat and Km remain unaffected. SDS-PAGE and Western blot analysis revealed exposure-dependent covalent aggregation with a simultaneous loss of native ALDH3A1 monomer. Biophysical studies demonstrated that both secondary and tertiary structure are disrupted after exposure to UV-light and also suggest specific chemical modifications, such as oxidation. CONCLUSIONS: Our results indicate that UV-light causes dramatic changes in the structure and function of ALDH3A1 protein. Significant activity loss is observed after brief UV-exposure and corresponds to the appearance of dimer and tetramer species. However, covalent aggregation does not entirely account for the observed activity loss, which leads us to conclude that there are multiple pathways of UV-induced ALDH3A1 modification.
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