title: Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle.
abstract: 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 (approximately 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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, Simons Building, 2095 Constant Avenue, Lawrence, KS 66047, USA.
authors.forename: R I
authors.initials: RI
authors.lastname: Viner
authors.forename: D A
authors.initials: DA
authors.lastname: Ferrington
authors.forename: T D
authors.initials: TD
authors.lastname: Williams
authors.forename: D J
authors.initials: DJ
authors.lastname: Bigelow
authors.forename: C
authors.initials: C
authors.lastname: Schöneich
mesh_terms: D000375:Aging / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018657:Muscle Fibers, Slow-Twitch / Q000201:enzymology; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011916:Rats, Inbred F344; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D009566:Nitrates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D010450:Endopeptidases; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D010100:Oxygen; D002118:Calcium