Ghost protein damage by peroxynitrite and its protection by melatonin
Braz. j. med. biol. res
; 33(1): 11-7, Jan. 2000. graf
Article
in English
| LILACS
| ID: lil-252251
Responsible library:
BR1.1
RESUMO
We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 µM ONOO-) disappearance at pH 7.4 of the main proteins spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ß-mercaptoethanol (2 percent) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50 percent at 250 µM ONOO-). However, a lack of effect of ß-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that ONOO--induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox® (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90 percent) at 500 µM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes
Full text:
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Collection:
International databases
Database:
LILACS
Main subject:
Free Radical Scavengers
/
Oxidants
/
Oxidative Stress
/
Cytoskeletal Proteins
/
Erythrocytes
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Melatonin
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Membrane Proteins
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Nitrates
/
Antioxidants
Limits:
Animals
Language:
English
Journal:
Braz. j. med. biol. res
Journal subject:
Biology
/
Medicine
Year:
2000
Document type:
Article