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.

Metal-catalyzed oxidation of histidine in human growth hormone. Mechanism, isotope effects, and inhibition by a mild denaturing alcohol.

Metal-catalyzed oxidation of proteins represents an important pathway of post-translational modification. We utilized human growth hormone (hGH), a protein with a well defined metal-binding site, to study the detailed mechanism of metal-catalyzed oxidation by ascorbate/Cu(II)/O2. Particularly His18 and His21 within the metal-binding site were oxidized, predominantly to 2-oxo-His with the incorporated oxygen originating from molecular oxygen, based on amino acid analysis, tryptic mapping, mass spectrometry, isotopic labeling, and 1H NMR. The anaerobic reduction of a hGH/Cu(II) mixture by ascorbate generated a hGH-Cu(I) complex with NMR spectral features different from those of native hGH and hGH/Cu(II). The anaerobic reaction of this hGH-Cu(I) complex with hydrogen peroxide resulted in the oxidation of His18 and His21, suggesting that a fraction of Cu(I) was bound at the metal-binding site of hGH. Site-specific oxidation of hGH required an intact metal-binding site and could largely (about 80%) be inhibited by the presence of >/=28% (v/v) 1-propanol which appears (i) to perturb the metal-binding site and (ii) to interact with a reactive oxygen species formed at the perturbed metal-binding site. The inhibition by 1-propanol-d7 (CD3CD2CD2OH) was significantly lower than that by 1-propanol-h7 with [residual hGH]1-propanol-h7/[residual hGH]1-propanol-d7 = 1.95 at 30% (v/v) 1-propanol, reflecting a kinetic isotope effect close to that for the reaction of a hydroxyl radical with Calpha-H/D bonds of methanol, suggesting the involvement of a hydroxyl radical-like species in the oxidation of His.