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Free fatty acids form peroxidase complexes with cyt c: role in mitochondrial oxidative stress and damage.

Kapralov-A; Vlasova-I; Belikova-N; Feng-W; Basova-L; Martin-J; Glumac-A; Bayir-H; Kagan-V
Toxicologist 2007 Mar; 96(1):61
Recently, we reported that negatively charged phospholipids- cardiolipin,phosphatidylserine- form strong complexes with cyt c in which the hemoprotein acts as a potent peroxidase capable of selective oxidation of respective phospholipids (Kagan et al., Nature Chem. Biol, 2005). Both electrostatic and hydrophobic interactions were found essential for the complexes formation. Free fatty acids (FFAs) carry both a negatively charged carboxy-group and long chain hydrophobic moiety. Because FFA accumulation occurs in membranes and biofluids in many dyslipidemias and other disease conditions, we hypothesized that they can stimulate peroxidase activity of cyt c. Here we report, that oleic acid effectively stimulated peroxidase activity of cyt c as evidenced by oxidation of three typical peroxidase substrates: Amplex Red, etoposide, and luminol. The peroxidase activation of cyt c occurred only at ratios of oleic acid/cyt exceeding 50:1. Generation of characteristic protein-derived radicals was confirmed by EPR spectroscopy of cyt c/oleic acid incubated with H2O2. Oligomerization of cyt c/FFA complexes was demonstrated by Western blotting. By utilization of immuno-spin trapping technique, we found the production of protein-immobilized DMPO nitrone adducts in the PAGE bands corresponding to oligomeric forms of cyt c. Two lines of evidence suggest interaction of cyt c with oleic acid unfolds the protein resulting in a greater accessibility of its heme: 1) in low temperature EPR spectra of heme-nitrosylated oleic acid/cyt c complexes, a signal of penta-coordinate heme-iron was detectable, and 2) increased fluorescence of tryptophan quenched in native enzyme by the heme moiety. Increased levels of cyt c-associated peroxidase activity were found in isolated mitochondria and in cells incubated with oleic acid suggesting that the complexes may function as a peroxidase in vivo.
Cell-alteration; Cell-damage; Cellular-reactions; Chemical-analysis; Chemical-composition; Chemical-indicators; Chemical-synthesis; Physiological-chemistry; Physiological-response; Physiological-effects; In-vivo-studies
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The Toxicologist. Society of Toxicology 46th Annual Meeting and ToxExpo, March 25-29, 2007, Charlotte, North Carolina
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University of Pittsburgh at Pittsburgh