F Wu1, G M Wang, N Raghavachari, M F Lou. 1. Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln 68583-0905, USA.
Abstract
PURPOSE: A new redox regulating enzyme, thioltransferase (TTase), has been found in the lens. The authors investigated whether TTase is also present in other ocular tissues. METHODS: Fresh enucleated bovine eyes were obtained from a local abattoir 4 hours after death. The eyes were processed immediately to remove corneal epithelial cells, conjunctiva, corneal endothelial cells, iris, ciliary body, lens epithelial cells, vitreous body, and retina. Other than conjunctiva and vitreous body, which were collected from a single eye, all other tissues were pooled from three bovine eyes. Each sample was homogenized in 0.1 M phosphate buffer, pH 7.4, and centrifuged at 10,000 g for 20 minutes, and the supernatant was assayed for TTase activity. Total RNA from each tissue sample was extracted and used for slot blot hybridization using cDNA from pig liver TTase with beta-actin as control. RESULTS: Among all the ocular tissues tested, iris showed the highest TTase activity (35 mU/mg protein) followed by conjunctiva, corneal epithelial cells, and corneal endothelial cells. Ciliary body, lens epithelial cells, and retina had moderate activity. No activity could be detected in vitreous body. The presence of this enzyme transcript in these ocular tissues was further confirmed by the positive slot blot hybridization with the pig liver TTase cDNA. Here again, iris showed the highest TTase mRNA expression, followed by ciliary body, lens epithelial cells, corneal endothelial cells, conjunctiva, retina, and corneal epithelial cells. The whole lens showed the lowest TTase mRNA expression, and no TTase mRNA was found in the vitreous body. CONCLUSIONS: TTase was found in most ocular tissues and was concentrated in the anterior segment of the eye. Highest activity was found in the iris, conjunctiva, corneal epithelial, and endothelial cells. TTase was absent in the vitreous body.
PURPOSE: A new redox regulating enzyme, thioltransferase (TTase), has been found in the lens. The authors investigated whether TTase is also present in other ocular tissues. METHODS: Fresh enucleated bovine eyes were obtained from a local abattoir 4 hours after death. The eyes were processed immediately to remove corneal epithelial cells, conjunctiva, corneal endothelial cells, iris, ciliary body, lens epithelial cells, vitreous body, and retina. Other than conjunctiva and vitreous body, which were collected from a single eye, all other tissues were pooled from three bovine eyes. Each sample was homogenized in 0.1 M phosphate buffer, pH 7.4, and centrifuged at 10,000 g for 20 minutes, and the supernatant was assayed for TTase activity. Total RNA from each tissue sample was extracted and used for slot blot hybridization using cDNA from pig liver TTase with beta-actin as control. RESULTS: Among all the ocular tissues tested, iris showed the highest TTase activity (35 mU/mg protein) followed by conjunctiva, corneal epithelial cells, and corneal endothelial cells. Ciliary body, lens epithelial cells, and retina had moderate activity. No activity could be detected in vitreous body. The presence of this enzyme transcript in these ocular tissues was further confirmed by the positive slot blot hybridization with the pig liver TTase cDNA. Here again, iris showed the highest TTase mRNA expression, followed by ciliary body, lens epithelial cells, corneal endothelial cells, conjunctiva, retina, and corneal epithelial cells. The whole lens showed the lowest TTase mRNA expression, and no TTase mRNA was found in the vitreous body. CONCLUSIONS:TTase was found in most ocular tissues and was concentrated in the anterior segment of the eye. Highest activity was found in the iris, conjunctiva, corneal epithelial, and endothelial cells. TTase was absent in the vitreous body.