In vivo aging of rat skeletal muscle sarcoplasmic reticulum Ca-ATPase. Chemical analysis and quantitative simulation by exposure to low levels of peroxyl radicals.
Sarcoplasmic reticulum (SR) Ca-ATPase of young adult (5 months) and aged (28 months) Fischer 344 male rat skeletal muscle was analyzed for posttranslational modifications as a result of biological aging and their potential functional consequences. The significant differences in the amino acid composition were a 6.8% lower content of sulfhydryl groups and a ca. 4% lower content of Arg residues of the Ca-ATPase from old as compared to young rats. Based on a total of 24 Cys residues the difference in protein thiols corresponds to a loss of 1.5 mol Cys/mol Ca-ATPase as a result of in vivo aging. The loss of Cys residues was not accompanied by a loss of enzyme activity though the 'aged' Ca-ATPase was more sensitive to heat inactivation, aggregation, and tryptic digestion. A comparison of the total sulfhydryl content of all SR proteins present revealed a 13% lower amount for SR vesicles isolated from aged rats. Compared to the alterations of Cys and Arg, there was only a slight and probably physiologically insignificant increase of protein carbonyls with aging, i.e. from 0.32 to 0.46 mol carbonyl groups per mol of Ca-ATPase. When SR vesicles from young rats were exposed to AAPH-derived peroxyl radicals, there was a loss of ca. 1.38 x 10(-4) M total SR sulfhydryl groups per 4 mg SR protein/ml (corresponding to ca. 25%) and a loss of 9.6 x 10(-5) M Ca-ATPase sulfhydryl groups (corresponding to ca. 31%) per 1.6 x 10(-5) M initiating peroxyl radicals, indicating that the stoichiometry of sulfhydryl oxidation was > or = 6 oxidized thiols per initiating AAPH-derived peroxyl radical. Besides Cys, the exposure to AAPH-derived radicals caused a slight loss of Ca-ATPase Arg, Met, and Ser residues. Most importantly, the SR Ca-ATPase exposed to this low concentration of peroxyl radicals displayed physical and functional properties quantitatively comparable to those of SR Ca-ATPase isolated from aged rats, i.e. no immediate loss of activity, increased susceptibility to heat inactivation, aggregation, and tryptic digestion. Moreover, a comparison of kinetically early tryptic fragments by HPLC-electrospray MS and N-terminal sequencing revealed that similar peptide fragments were produced from 'aged' and AAPH-oxidized Ca-ATPase which were not (or kinetically significantly later) generated from the 'young' Ca-ATPase, suggesting some conformational changes of the Ca-ATPase as a result of aging and AAPH-exposure. All except one of these peptides originated from locations remote from the nucleotide-binding and calcium-binding sites. The latter results suggest that aging and AAPH-exposure may target similar Cys residues, mainly at locations remote from the nucleotide-binding and calcium-binding sites, rationalizing the fact that Cys oxidation did not immediately cause inactivation of the Ca-ATPase. Our results provide a quantitative estimate of a net concentration of reactive oxygen species, here peroxyl radicals, which induces physical and chemical alterations of the SR Ca-ATPase quantitatively comparable to those induced by in vivo aging.
Viner
R I
RI
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
Ferrington
D A
DA
Aced
G I
GI
Miller-Schlyer
M
M
Bigelow
D J
DJ
Schöneich
C
C
D000375:Aging / Q000378:metabolism*; D000578:Amidines / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D005609:Free Radicals / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008297:Male; D024510:Muscle Development; D018482:Muscle, Skeletal / Q000201:enzymology* / Q000254:growth & development; D009153:Mutagens / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010545:Peroxides / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011916:Rats, Inbred F344; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013816:Thermodynamics; D014357:Trypsin
D000578:Amidines; D000596:Amino Acids; D005609:Free Radicals; D009153:Mutagens; D010446:Peptide Fragments; D010545:Peroxides; D013438:Sulfhydryl Compounds; C049375:perhydroxyl radical; C046728:2,2'-azobis(2-amidinopropane); D014357:Trypsin; D000252:Calcium-Transporting ATPases