Veterinary and Biomedical Sciences, Department of


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A THESIS Presented to the Faculty of the Graduate College at the University of Nebraska in Partial Fulfillment of Requirements for the Degree of Master of Science (Major: Veterinary Science) under the Supervision of Professor Marjorie F. Lou; Lincoln, Nebraska, USA; August, 2012

Copyright 2012 Bijaya Prasad Upadhyaya


Glutaredoxin 2 (Grx2), a thiol-regulating enzyme of oxidoreductase family and a mitochondrial isozyme of glutaredoxin 1, was discovered 11 years ago in our laboratory. Grx2 is present in the lens where it shows dethiolase, peroxidase, and ascorbate recycling activities. Recently, Grx2 has also been identified to protect the mitochondrial electron transport system with anti-apoptotic function. Since other eye tissues besides the lens are rich in mitochondria and are very sensitive to oxidative stress, we speculate the presence of Grx2 therein as an important redox regulator. This study is to investigate the expression and distribution of Grx2 in ocular tissues using porcine eye as a model.

Fresh enucleated porcine eyes obtained from a local abattoir were immediately dissected into cornea, iris, the lens, vitreous humor, ciliary body, retina, and optic nerve; frozen in dry ice; and stored at -80oC. Each sample with 3 tissues pooled was homogenized in 1.0 ml of ice-cold 10 mM HEPES buffer (pH 7.2) containing 225 mM D-mannitol, 65 mM sucrose and 1mM EGTA in a glass-to-glass homogenizer, followed by a series of centrifugations to remove tissue debris while isolating mitochondrial fraction. Mitochondrial sample proteins of each sample were separated by 12% SDS-PAGE, followed by Western blot analysis to detect Grx2 by a specific Grx2 antibody. The same tissue sample with 5mg/ml mitochondrial protein was completely dissolved in 1% lauryl maltoside, and measured for Grx2 enzyme activity following the published procedure.

Western blots showed the expression of Grx2 in all the tested ocular tissues, except vitreous humor. Relative expression of Grx2 to the positive control, mouse mitochondrial liver homogenate, revealed that the ciliary body had the highest expression ratio (26.64), followed by the retina (11.92), and optic nerve (8.60). The lens had the lowest expression ratio of 0.75, while the vitreous humor did not show any Grx2 positive band. Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.89 mU/mg protein), closely followed by ciliary body (3.10 mU/mg), lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Vitreous humor had no Grx2 activity.

In conclusion, Grx2 was found in all porcine ocular tissues except for vitreous humor. The Grx2 protein expression level was higher in eye tissues rich in mitochondria (ciliary body and retina), corroborating with their high Grx2 activity. The rich presence of Grx2 in these tissues is consistent with their known sensitivity to oxidative stress.

Advisor: Marjorie F. Lou