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

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Abstract
We have examined the oxidative sensitivity of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum (SR) membranes, exposing isolated SR membranes to the thermolabile water soluble free radical initiator, 2,2‘-azobis(2-amidinopropane) dihydrochloride (AAPH). Incubation with up to 702 μM AAPH-derived radicals results in a concentration- and time-dependent inhibition of calcium-dependent ATPase activity correlating with the loss of monomeric Ca2+-ATPase polypeptides, and the concomitant appearance of higher molecular weight species. However, no oxidant-induced protein fragmentation is detected. The observed formation of oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPase cross-links, as well as intramolecular cross-links. The oxidation of sulfhydryl groups to disulfides as another possible source of intermolecular cross-links has been ruled out after examination of SDS−PAGE performed under both reducing and non-reducing conditions. Exposure of the SR membranes to AAPH-derived radical species results in a small degree of lipid peroxidation that is not correlated with enzyme inactivation, suggesting that modification of membrane-spanning peptides is not related to enzyme inactivation. Six cytoplasmic peptides have been identified that are modified by exposure to AAPH or, alternatively, to hydrogen peroxide, suggesting that these regions of the Ca2+-ATPase are generally sensitive to oxidants. These oxidized peptides were identified after separation by reversed-phase HPLC followed by N-terminal sequencing and amino acid analysis as corresponding to the following sequences of the Ca2+-ATPase: (i) Glu121 to Lys128, (ii) His190 to Lys218, (iii) Asn330 to Lys352, (iv) Gly432 to Lys436, (v) Glu551 to Arg604, and (vi) Glu657 to Arg671. The Glu551 to Arg604 peptide, located within the nucleotide binding domain, was found to participate in the formation of intermolecular bityrosine cross-links with the identical Glu551 to Arg604 peptide from a neighboring Ca2+-ATPase polypeptide chain.