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Xiaoli Tian, Bijaya Upadhyaya, Hongli Wu, Marjorie Lou; EXPRESSION AND DISTRIBUTION OF THIOL-REGULATING ENZYME GLUTAREDOXIN 2 (Grx2) IN PORCINE OCULAR TISSUES. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4963. doi: https://doi.org/.
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Glutaredoxin 2 (Grx2) is a mitochondrial isozyme of glutaredoxin 1, and ubiquitously present in all life forms and plays an important role in maintaining redox homeostasis in the cells. Grx2 is found in the lens where its multiple activities of dethiolase, peroxidase, and dehydro- ascorbate reductase can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, we investigate the presence of Grx2 in various ocular tissues.
Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized in 1.0 ml 10 mM HEPES buffer (pH 7.2), followed by mitochondrial isolation. The presence of Grx2 protein in each mitochondrial fraction was analyzed using Western blotting with anti-Grx2 antibody; while the Grx2 activity was measured following the published procedure. Some freshly dissected porcine eyes were used to measure Grx2 mRNA expression by RT-PCR analysis with GAPDH as the control. The Grx2-rich mouse liver was used as a positive control for the above analyses.
Western blot analysis showed that Grx2 was present in all the tested ocular tissues, except vitreous humor. Relative expression of Grx2 protein in each ocular tissue to mouse liver, revealed that the ciliary body had the highest expression ratio (26.6), followed by the retina (11.9), and optic nerve (8.6). The lens had the lowest expression ratio of 0.75, while the vitreous humor had none. Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Vitreous humor had no Grx2 activity. Grx2 gene expression in these ocular tissues was further confirmed by GRX2 cDNA. Ciliary body showed the highest Grx2 mRNA expression, followed by retina, optical nerve, cornea and iris. The expression of Grx2 mRNA was low in the lens while none in the vitreous humor.
Grx2 was found in most porcine ocular tissues except vitreous humor. The Grx2 protein expression level was higher in eye tissues rich in mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is consistent with their known sensitivity to oxidative stress.
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