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G.–S. Wu, N.A. Rao; Cytochrome c is the Major Nitrated Protein in Mitochondria in the Early Phase of Experimental Autoimmune Uveoretinitis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):809.
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We previously found that three proteins were significantly nitrated in the whole retinal extracts in the early phase of experimental autoimmune uveitis (EAU). All three proteins are involved in major mitochondrial functions, including energy production, metabolism, and protein import from cytosol (IOVS 2005;46:2271). In this study, we sought to define in particular the in vivo tyrosine–nitrated products that originate in the mitochondria by isolating the mitochondria from the early EAU retina.
EAU was induced in Lewis rats with bovine S–antigen. Mitochondria were isolated from the retinas on days 0, 5 and 10 postimmunization by sucrose gradient centrifugation. Day 15 is the peak of inflammation in this system. Sixteen retinas were harvested for each time period, and eight retinas were combined as one determination. Mitochondria were sonicated without detergent and separated into two fractions: soluble proteins and membranes. Immunoblots with anti–nitrotyrosine were used to detect tyrosine–nitrated proteins and anti–cytochrome c (cyto c) was used to confirm cyto c identification. Membrane fraction was further solubilized before immunoblotting.
Coomassie blue staining revealed numerous bands in both soluble protein and membrane fractions for all time periods. In immunoblots, soluble protein fraction on both days 5 and 10 displayed one major nitrated band at 15 kDa, and this band was absent on day 0. Low–abundance bands were seen in the 36–50 kDa region; but some of these bands were also present in day 0 mitochondria. The 15 kDa band was subsequently identified by immunoblots probed with anti–rat cyto c. In membrane fractions, no distinctive nitrotyrosine positive band was seen and only faint cyto c bands were visible in all three time periods.
Cytochrome c embedded in the inner membrane of mitochondria is an electron transporter and faces the intermembrane space. This physical situation appears to make cyto c vulnerable to the gradient of peroxynitrite generated in the mitochondrial intermembrane space. Therefore, retinal tissue damage through mitochondrial nitration has already been set forth long before the arrival of inflammatory infiltrates.
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