The accumulation of covalently modified proteins is an important hallmark of biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification of specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, Xu and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significant age-dependent change in ATP-supported Ca2+-uptake activity for slow-twitch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-muscle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a from fast-twitch and slow-twitch muscle respectively. We find a significant age-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantly slow-twitch, but not from fast-twitch, muscles. Western immunoblotting and amino acid analysis demonstrate that, selectively, the SERCA2a isoform progressively accumulates a significant amount of nitrotyrosine with age (≈ 3.5±0.7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isoforms suffer an age-dependent loss of reduced cysteine which is, however, functionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O2) results in the selective nitration only of the SERCA2a, suggesting that ONOO- may be the source of the nitrating agent in vivo. A correlation of the SR Ca2+-ATPase activity and covalent protein modifications in vitro and in vivo suggests that tyrosine nitration may affect the Ca2+-ATPase activity. By means of partial and complete proteolytic digestion of purified SERCA2a with trypsin or Staphylococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC-electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dependent tyrosine nitration to the sequence Tyr294-Tyr295 in the M4-M8 transmembrane domain of the SERCA2a, close to sites essential for Ca2+ translocation.
- nitric oxide
- thiol modification
- The Biochemical Society, London © 1999