Activation of Microsomal GlutathioneS-Transferase by Peroxynitrite

Abstract

Peroxynitrite (ONOO Ϫ ) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO Ϫ on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4-to 5-fold by 2 mM ONOO Ϫ ; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO Ϫ resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO Ϫ -treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO Ϫ formation.

Urinary felinine excretion in intact male cats is increased by dietary cystine

“…After a 9 d period during which the animals adapted to the metabolism cages (days 1-9), urine was collected quantitatively for 5 d (days [10][11][12][13][14] and pooled per cat. Thereafter, the cats received the moist HP diet (Table 1) for a further 9 d (days 15-23) before urine was again collected for 5 d (days [24][25][26][27][28] and pooled per cat. The calculated metabolisable energy (ME) of both the LP and the HP diets was the same (20·1 kJ (4·8 kcal)/g DM) as determined using 14·6, 35·6 and 14·6 kJ/g DM (3·5, 8·5 and 3·5 kcal/g DM), respectively for protein, fat and carbohydrate.…”

“…Urine was collected quantitatively for 5 d and pooled for each cat, after which (day 6), group A received the LP diet supplemented with 0·93 % L-cystine (LPc; Table 1) on days 6-15 and then the LP diet from day 16 to day 23. On day 6, group B received the LP diet supplemented with cystine, arginine and three (glutamic acid, glycine and proline) dispensable amino acids until day 16 after which they received the LP diet supplemented with 0·93 % L-cystine for 10 d (days [17][18][19][20][21][22][23][24][25][26]. From day 27 until day 34, the cats in group B were again fed the LP diet supplemented with cystine, dispensable amino acids and arginine (LPc þ D þ arg; Table 1).…”

Nitration of Tyrosine 92 Mediates the Activation of Rat Microsomal Glutathione S-Transferase by Peroxynitrite

“…The ONOO Ϫ -induced activation of MGST1 was associated with tyrosine nitration, polymer formation, and protein fragmentation but not protein S-oxidation (3). This increase in enzyme activity by ONOO Ϫ is unusual, since tyrosine nitration of proteins is almost always associated with a loss of function.…”

“…Activation of MGST1 also occurs after exposure to reactive nitrogen species (RNS), including nitric oxide (NO) donors and peroxynitrite (ONOO Ϫ ) (3,4), in contrast to the cytosolic GSTs, which are inhibited by RNS (5). The ONOO Ϫ -induced activation of MGST1 was associated with tyrosine nitration, polymer formation, and protein fragmentation but not protein S-oxidation (3).…”

“…In contrast to the cytosolic GSTs, an important characteristic of microsomal GST1 is that the enzyme can be activated by various treatments, including limited proteolysis, heating, radiation, and exposure to sulfhydryl modifying reagents and reactive oxygen species. In addition, since MGST1 possesses glutathione peroxidase activity, it has been suggested that this enzyme may play a protective role under conditions of oxidative stress (2,3).…”