Nitration of specific tyrosines in FoF1 ATP synthase and activity loss in aging

Abstract

It has been reported that C-nitration of proteins occurs under nitrative/oxidative stress; however, its role in pathophysiological situations is not fully understood. In this study, we determined that nitration of Tyr(345) and Tyr(368) in the beta-subunit of the mitochondrial F(o)F(1)-ATPase is a major target for nitrative stress in rat liver under in vivo conditions. The chemical characteristics of these Tyr make them suitable for a facilitated nitration (solvent accessibility, consensus sequence, and pK(a)). Moreover, beta-subunit nitration increased significantly with the age of the rats (from 4 to 80 weeks old) and correlated with decreased ATP hydrolysis and synthesis rates. Although its affinity for ATP binding was unchanged, maximal ATPase activity decreased between young and old rats by a factor of two. These changes directly impacted the available ATP concentration in vivo, and it was expected that they would affect multiple cellular ATP-dependent processes. For instance, at least 50% of available [ATP] in the liver of older rats would have to be committed to sustain maximal Na(+)-K(+)-ATPase activity, whereas only 30% would be required for young rats. If this requirement was not fulfilled, the osmoregulation and Na(+)-nutrient cotransport in liver of older rats would be compromised. On the basis of our studies, we propose that targeted nitration of the beta-subunit is an early marker for nitrative stress and aging.

Fig. 1.

Immunoprecipitation of nitrated proteins from mitochondria. Mitochondrial fractions [total (T), inner membrane (IM), contact sites (CS)] were immunoprecipitated with nitrotyrosine (A and B) or β-subunit antibodies (C and D). Western blots of the immunoprecipitated fractions were then probed for either nitrotyrosine (A and C) or β-subunit (B and D) antibodies. Blots in A and C were blocked with nonfat dry milk (NFDM) with the addition of F(Ab)₂, whereas blots in B and D were blocked with NFDM only. This control was done to identify the band appearing at 63 kDa as the heavy chain of immunoglobulins from the immunoprecipitation procedure. E: l-argininne-supplemented rat liver mitochondria isolated from young rats were incubated for 0, 5, 15, 30, and 60 min (●). Samples at each time point were separated by two-dimensional gels, probed for nitrotyrosine, and then stripped and probed for the β-subunit by using Western blots. Control experiments were performed by incubating mitochondria with N^G-monomethyl-l-arginine (○), an inhibitor of nitric oxide (NO) synthase. MWM, molecular weight markers.

Fig. 2.

Alignment of rat ATP synthase-α and -β chains. The sequences for these subunits of ATP synthase were downloaded from the SWISSPROT database and aligned with CLUSTALW. The alignment exhibited 24% identities (fully conserved residues: 100/401; dark gray) and 42% positive residues (conservation of strong groups: 174/401; light gray). *Tyr³⁴⁵ and Tyr³⁶⁸.

Fig. 3.

Activity of liver ATPase in young, adult, and old animals. The activity of ATPase was evaluated (following the procedure described in experimental procedures) in rat liver mitochondria from young (▲), adult (○), and old rats (●).

Fig. 4.

ATP concentrations, Na⁺-K⁺-ATPase activity, and oxygen uptake of rat liver slices incubated in the presence of different concentrations of potassium cyanide (KCN). Liver slices (0.4 μm) from young (○ and ☐) and old (● and ■) rats were incubated in Krebs-Henseleit buffer for 60 min at 30°C. After the slices in this medium were incubated (and after the medium was changed several times), the buffer was supplemented with various amounts of [KCN]. Oxygen consumption was evaluated with a Clark-type electrode. Tissue slice incubations, [ATP] (☐ and ■), and Na⁺ extrusion used to evaluate Na⁺-K⁺-ATPase activity (○ and ●) were performed as described previously by others (4, 71).