MECHANISMS OF PROTECTION AGAINST PEROXIDATIVE DAMAGE

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We have previously reported that vitamin E is enzymatically regenerated by reduced glutathione (GSH) in rat liver microsomes and later in human liver microsomes. Recently several other groups have provided evidence in support of this hypothesis; however, the nature enzyme(s) involved in GSH-dependent protection against microsomal lipid peroxidation have not been fully characterized. The primary objective of the proposed research is to delineate the molecular mechanisms underling the vitamin E-dependent inhibition of lipid peroxidation by reduced glutathione in rat liver microsomes. Our central hypothesis is that there are at least two mechanisms, one appears to be vitamin E-dependent and the other is vitamin E-independent, which are responsible for the inhibition of lipid peroxidation in rat liver microsomes that exhibit an absolute requirement to GSH and/or GSSG for their action. We propose that one mechanism is responsible for the GSH-dependent enzymatic regeneration of alpha- tocopherol (alpha-TH) from the alpha-tocopheroxyl radical during oxidative stress. The second mechanism involves a protein which is modified by thiol:disulfide exchange, that may function independent of (alpha-TH to reduce lipid hydroperoxides that are formed during lipid peroxidation. Our hypothesis therefore, is based on the premise that protection against membrane lipid peroxidation is associated with specific proteins that are regulated by physiologically important sulfhydryls and disulfides. The specific aims of the proposed research are to 1) characterize the nature of the GSH-dependent protein involved in the regeneration of (x-TH in rat liver microsomes, and to investigate the mechanism of interaction of GSH, (alpha-TH, and the alpha-TH regenerating protein both in vivo and in vitro; 2) examine the GSH/GSSG-dependent protein involved in the protection against lipid peroxidation in the liver; 3) study the effects of physiological, thiol:disulfide redox couples in addition to GSH:GSSG in the inhibition of membrane lipid peroxidation; and 4) examine the temporal relationships among alpaha-TH, protein SH, mixed protein disulfide formation and lipid peroxidation. Experimental procedures to include molecular probes such as antibodies and cDNA will be generated for the purified protein exhibiting GSH-dependent activity towards the reduction of the alpha-tocopheroxyl radical as well as for the membrane GST isoenzyme(s) that may be regulated by thiol:disulfide exchange. Multilamellar liposomal model systems will be employed to investigate details of the mechanism of inhibition of lipid peroxidation by the purified proteins. Western blot analysis and RNA blot analysis as well as nuclear run-on transcriptional assays will be performed to determine the effects of altered vitamin E nutrition on the expression of the GSH-dependent proteins involved in the inhibition of lipid peroxidation. New information derived from this proposal should enhance our understanding on the control of free radical-mediated damage to biological membranes.