Identification of Oxidation-Sensitive Peptides within the Cytoplasmic Domain of the Sarcoplasmic Reticulum Ca2+-ATPase

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• 作者：Rosa I. Viner ; Arkadi G. Krainev ; Todd D. Williams ; Christian Schö ; neich ; and Diana J. Bigelow
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：June 24, 1997
• 年：1997
• 卷：36
• 期：25
• 页码：7706 - 7716
• 全文大小：529K
• 年卷期：v.36,no.25(June 24, 1997)
• ISSN：1520-4995

文摘

We have examined the oxidative sensitivity of theCa²⁺-ATPase of skeletal muscle sarcoplasmicreticulum (SR) membranes, exposing isolated SR membranes to thethermolabile water soluble free radicalinitiator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH).Incubation with up to 702 ges/entities/mgr.gif">M AAPH-derived radicals results in a concentration- and time-dependentinhibition of calcium-dependent ATPaseactivity correlating with the loss of monomericCa²⁺-ATPase polypeptides, and the concomitantappearanceof higher molecular weight species. However, no oxidant-inducedprotein fragmentation is detected. Theobserved formation of oxidant-induced bityrosine accounts for theintermolecular Ca²⁺-ATPase cross-links, as well as intramolecular cross-links. The oxidation ofsulfhydryl groups to disulfides as anotherpossible source of intermolecular cross-links has been ruled out afterexamination of SDS-PAGE performedunder both reducing and non-reducing conditions. Exposure of theSR membranes to AAPH-derivedradical species results in a small degree of lipid peroxidation that isnot correlated with enzyme inactivation,suggesting that modification of membrane-spanning peptides is notrelated to enzyme inactivation. Sixcytoplasmic peptides have been identified that are modified by exposureto AAPH or, alternatively, tohydrogen peroxide, suggesting that these regions of theCa²⁺-ATPase are generally sensitive tooxidants.These oxidized peptides were identified after separation byreversed-phase HPLC followed by N-terminalsequencing and amino acid analysis as corresponding to the followingsequences of the Ca²⁺-ATPase:(i) Glu₁₂₁ to Lys₁₂₈, (ii) His₁₉₀to Lys₂₁₈, (iii) Asn₃₃₀ to Lys₃₅₂,(iv) Gly₄₃₂ to Lys₄₃₆, (v) Glu₅₅₁to Arg₆₀₄,and (vi) Glu₆₅₇ to Arg₆₇₁. TheGlu₅₅₁ to Arg₆₀₄ peptide, located within thenucleotide binding domain,was found to participate in the formation of intermolecular bityrosinecross-links with the identical Glu₅₅₁to Arg₆₀₄ peptide from a neighboringCa²⁺-ATPase polypeptide chain.