Protein modification during biological aging: selective tyrosine nitrationof the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle

The accumulation of covalently modified proteins is an important hallmark o f biological aging, but relatively few studies have addressed the detailed molecular-chemical changes and processes responsible for the modification o f specific protein targets. Recently, Narayanan et al. [Narayanan, Jones, X u and Yu (1996) Am. J. Physiol. 271, C1032-C1040] reported that the effects of aging on skeletal-muscle function are muscle-specific, with a significa nt age-dependent change in ATP-supported Ca2+-uptake activity for slow-twit ch but not for fast-twitch muscle. Here we have characterized in detail the age-dependent functional and chemical modifications of the rat skeletal-mu scle sarcoplasmic-reticulum (SR) Ca2+-ATPase isoforms SERCA1 and SERCA2a fr om fast-twitch and slow-twitch muscle respectively. We find a significant a ge-dependent loss in the Ca2+-ATPase activity (26% relative to Ca2+-ATPase content) and Ca2+-uptake rate specifically in SR isolated from predominantl y slow-twitch, but not from fast-twitch, muscles. Western immunoblotting an d amino acid analysis demonstrate that, selectively, the SERCA2a isoform pr ogressively accumulates a significant amount of nitro-tyrosine with age (ap proximate to 3.5 +/- 0.7 mol/mol of SR Ca2+-ATPase). Both Ca2+-ATPase isofo rms suffer an age-dependent loss of reduced cysteine which is, however, fun ctionally insignificant. In vitro, the incubation of fast- and slow-twitch muscle SR with peroxynitrite (ONOO-) (but not NO/O-2) results in the select ive 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 activit y and covalent protein modifications in vitro and in vivo suggests that tyr osine nitration may affect the Ca2+-ATPase activity. By means of partial an d complete proteolytic digestion of purified SERCA2a with trypsin or Staphy lococcus aureus V8 protease, followed by Western-blot, amino acid and HPLC- electrospray-MS (ESI-MS) analysis, we localized a large part of the age-dep endent tyrosine nitration to the sequence Tyr(294)-Tyr(295) in the M4-M8 tr ansmembrane domain of the SERCA2a, close to sites essential for Ca2+ transl ocation.