Nitration/S-nitrosation of proteins by peroxynitrite-treatment and subsequent modification by glutathione S-transferase and glutathione peroxidase.
In various peroxynitrite (PN)-treated proteins, the formations of stable 3-nitrotyrosine (nitration) and labile S-nitrosocysteine (S-nitrosation) were observed by employing rapid Western blot in 6 h. The steps of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and membrane-blotting were performed at 4 degrees C. It was noted that the intensity of immunoreactive bands specific for anti-nitrotyrosine was stronger than that specific for anti-S-nitrosocysteine. Additionally, the intensity was in the manner of a dose-dependency of PN. Nitration/S-nitrosation were formed in the following treated proteins, including bovine serum albumin (BSA), DNase-1, ceruloplasmin, catalase and hemoglobin (Hb). The incubation of PN-pretreated hemoglobin with 1 mM reduced glutathione (GSH) did not change immunoreactivity significantly. However, the addition of glutathione S-transferase (GST) or glutathione peroxidase (GPX) to the above incubation mixture, resulted in decreased immunoreactivity, suggesting GSH may form a transition complex with PN-pretreated hemoglobin and/or partially reduce/modify the treated hemoglobin, thereby increasing the accessibility for the subsequent modification by GST or GPX. Such decreased immunoreactivity indicates that nitrotyrosine and S-nitrosocysteine of treated hemoglobin was, indeed, further modified via (a) converting -NO2 to -NH2 in tyrosine residues, (b) denitrating -NO2 directly/indirectly in tyrosine residues, and/or (c) changing -S-NO to -SH in cysteine residues, or denitrosation. The findings imply similar enzymatic modifications of proteins may also occur in vivo, and therefore play a pivotal role in the NO-related cellular signaling cascade(s).
Kuo
Wu-Nan
WN
Division of Science and Mathematics, Bethune-Cookman College, Daytona Beach, FL 32114, USA.
Kocis
Joseph M
JM
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000494:pharmacology*; D006454:Hemoglobins / Q000187:drug effects* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006736:Horses; D066298:In Vitro Techniques; D051379:Mice; D015538:Nitrosation; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D026403:S-Nitrosothiols / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D006454:Hemoglobins; D006657:Histones; D026403:S-Nitrosothiols; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C021315:S-nitrosocysteine; D005979:Glutathione Peroxidase; D005982:Glutathione Transferase; D005978:Glutathione; D003545:Cysteine