An activity in rat tissues that modifies nitrotyrosine-containing proteins

Abstract

Homogenates from rat spleen and lung could modify nitrotyrosine-containing BSA. With incubation, nitrotyrosine-containing BSA lost its epitope to a monoclonal antibody that selectively recognized nitrotyrosine-containing proteins. In the presence of protease inhibitors, the loss of the nitrotyrosine epitope occurred without protein degradation and hydrolysis. This activity was found in supernatant but not particulate fractions of spleen homogenates. The factor was heat labile, was sensitive to trypsin treatment, and was retained after passage through a membrane with a 10-kDa retention. The activity was time-and protein-concentration dependent. The activity increased about 2-fold in spleen extracts with endotoxin (bacterial lipopolysaccharide) treatment of animals, suggesting that the activity is inducible or regulatable. Other nitrotyrosine-containing proteins also served as substrates, while free nitrotyrosine and some endogenous nitrotyrosine-containing proteins in tissue extracts were poor substrates. Although the product and possible cofactors for this reaction have not yet been identified, this activity may be a ''nitrotyrosine denitrase'' that reverses protein nitration and, thus, decreases peroxynitrite toxicity. This activity was not observed in homogenates from rat liver or kidney, suggesting that there may also be some tissue specificity for the apparent denitrase activity.

First evidence for an LDL‐ and HDL‐associated nitratase activity that denitrates albumin‐bound nitrotyrosine—Physiological consequences

“…Characteristics of the present nitratase activity are in total agreement with those found in tissues and mitochondria by Western immunoblot analyses (11)(12)(13)31). It has been hypothesized (13) that denitration of one nitrotyrosine residue is described by the following reaction: Protein-Tyr-NO 2 …”

“…They also lead us to establish that the present nitratase is actually a protein nitratase (or an albumin nitratase), as it was not active towards free nitrotyrosine in line with (31). Whether the present nitratase has a strict substrate specificity or not (i.e., specifically needs albumin as substrate), however, remain to be explored.…”

Superoxide as a Messenger of Endothelial Function

“…This suggests the "reactivation" of the enzyme by a mechanism apparently independent of a de novo synthesis of the enzyme. First reports for such a concept came from the laboratory of Murad (Kamisaki et al, 1998). He discovered that the nitration of BSA was reduced after incubation of the protein together with NADPH and spleen homogenate from endotoxin treated rats.…”

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

“…Formation of protein 3-nitrotyrosine has been detected in a number of disease states, but the functions of this protein modification remain unclear. Although there is some suggestion of reversibility, there is no convincing molecular evidence for a eucaryotic denitrase (Gow et al, 1996;Kamisaki et al, 1998). There appears to be some selectivity in the nitration, since not all proteins in a tissue under analysis are nitrated.…”