Peptide-based Antibodies against Glutathione-binding Domains Suppress Superoxide Production Mediated by Mitochondrial Complex I
oxidative stress; Biochemistry & Molecular Biology; nitric-oxide; proteins; site; generation; s-nitrosylation; postischemic heart; radical formation; bovine heart-mitochondria; nadh-ubiquinone oxidoreductase
Complex I (NQR) is a critical site of superoxide (O-2(radical anion)) production and the major host of redox protein thiols in mitochondria. In response to oxidative stress, NQR-derived protein thiols at the 51- and 75-kDa subunits are known to be reversibly S-glutathionylated. Although several glutathionylated domains from NQR 51 and 75 kDa have been identified, their roles in the regulatory functions remain to be explored. To gain further insights into protein S-glutathionylation of complex I, we used two peptides of S-glutathionylated domain ((200)GAGAYI (C) under bar GEETALIESIEGK(219) of 51-kDa protein and (VDSDTL)-V-361 (C) under bar TEEVFPTAGAGTDLR(382) of 75-kDa protein) as chimeric epitopes incorporating a "promiscuous" T-cell epitope to generate two polyclonal antibodies, AbGSCA206 and AbGSCB367. Binding of AbGSCA206 and AbGSCB367 inhibited NQR-mediated O-2(radical anion). generation by 37 and 57%, as measured by EPR spin-trapping. To further provide an appropriate control, two peptides of non-glutathionylated domain ((21)SGDTTAPKKTSFGSLKDFDR(40) of 51-kDa peptide and (100)WNILTNSEKTKKAREGVMEFL(120) of 75-kDa peptide) were synthesized as chimeric epitopes to generate two polyclonal antibodies, Ab51 and Ab75. Binding of A51 did not affect NQR-mediated O-2(radical anion) generation to a significant level. However, binding of Ab75 inhibited NQR- mediated O-2(radical anion) generation by 35%. None of AbGSCA206, AbGSCB367, Ab51, or Ab75 showed an inhibitory effect on the electron transfer activity of NQR, suggesting that antibody binding to the glutathione-binding domain decreased electron leakage from the hydrophilic domain of NQR. When heart tissue homogenates were immunoprecipitated with Ab51 or Ab75 and probed with an antibody against glutathione, protein S-glutathionylation was enhanced in post-ischemic myocardium at the NQR 51-kDa subunit, but not at the 75-kDa subunit, indicating that the 51-kDa subunit of flavin subcomplex is more sensitive to oxidative stress resulting from myocardial infarction.
Chen J F; Chen C L; Rawale S; Chen C A; Zweier J L; Kaumaya P T P; Chen Y R
Journal of Biological Chemistry
2010
2010-01
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1074/jbc.M109.056846" target="_blank" rel="noreferrer noopener">10.1074/jbc.M109.056846</a>
Oxidative damage of DNA by chromium(V) complexes: relative importance of base versus sugar oxidation
Biochemistry & Molecular Biology; in-vitro; nucleic-acids; abstraction; aqueous-solution; carcinogen chromium(vi); hydrogen; molecular-oxygen; radical formation; redox potentials; singlet oxygen; strand breaks
Chromium(V)-mediated oxidative damage of deoxyribonucleic acids was investigated at neutral pH in aqueous solution by utilizing bis(2-ethyl-2-hydroxybutanato)oxochromate(V) (I) and bis(hydroxyethyl)amino-tris(hydroxymethyl)methane)oxochromate(V) (II). Single-stranded and double-stranded (ds) calf thymus and human placenta DNA, as well as two oligomers, 5'-GATCTAGTAGGAGGACAAATAGTGTPTG-3' and 5'-GATCCAAGCAAACACTATTTGTCCTCCTACTA-3', were reacted with the chromium(V) complexes. Most products were separated and characterized by chromatographic and spectroscopic methods, Polyacrylamide gel electrophoresis experiments reveal more damage at G sites in comparison to other bases. Three primary oxidation products, 5-methylene-2-furanone (5-MF), furfural and 8-oxo-2'-deoxyguanosine, were characterized, A minor product, which appears to be thymine propenal, was also observed. The dsDNA produces more furfural than furanone, The formation of these two products resulted from hydrogen abstraction dr hydride transfer from C1' and C5' positions of the ribose to the oxo-chromium(V) center. Since no enhancements of these products (except propenal) were observed in the presence of oxygen, mechanisms pertaining to the participation of activated oxygen species may be ruled out. The oxidation of the G base is most likely associated with an oxygen atom transfer from the oxo-metallates to the double bond between C8 and N7 of the purine ring. The formation of the propenal may be associated with an oxygen-activated species, since a marginal enhancement of this product was observed in the presence of oxygen, The formation of furfural in higher abundance over 5-MF for dsDNA was attributed to the ease of hydrogen abstraction (or hydride transfer) from the C5' compared to C1' position of the ribose within a Cr(V)-DNA intermediate in which the metal center is bound to the phosphate diester moiety.
Bose R N; Moghaddas S; Mazzer P A; Dudones L P; Joudah L; Stroup D
Nucleic Acids Research
1999
1999-05
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1093/nar/27.10.2219" target="_blank" rel="noreferrer noopener">10.1093/nar/27.10.2219</a>