IDENTIFICATION OF OXIDATION-SENSITIVE PEPTIDES WITHIN THE CYTOPLASMICDOMAIN OF THE SARCOPLASMIC-RETICULUM CA2-ATPASE()

We have examined the oxidative sensitivity of the Ca2+-ATPase of skele tal 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 u p to 702 mu M AAPH-derived radicals results in a concentration- and ti me-dependent inhibition of calcium-dependent ATPase activity correlati ng with the loss of monomeric Ca2+-ATPase polypeptides, and the concom itant appearance of higher molecular weight species. However, no oxida nt-induced protein fragmentation is detected. The observed formation o f oxidant-induced bityrosine accounts for the intermolecular Ca2+-ATPa se cross-links, as well as intramolecular cross-links. The oxidation o f sulfhydryl groups to disulfides as another possible source of interm olecular 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 d egree of lipid peroxidation that is not correlated with enzyme inactiv ation, suggesting that modification of membrane-spanning peptides is n ot 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 a re generally sensitive to oxidants. These oxidized peptides were ident ified after separation by reversed-phase HPLC followed by N-terminal s equencing and amino acid analysis as corresponding to the following se quences of the Ca2+-ATPase: (i) Glu(121) to Lys(128), (ii) His(190) to Lys(218), (iii) Asn(330) to Lys(352), (iv) Gly(432) to Lys(436), (v) Glu(551) to Arg(604), and (vi) Glu(657) to Arg(671). The Glu(551) to A rg(604) peptide, located within the nucleotide binding domain, was fou nd to participate in the formation of intermolecular bityrosine cross- links with the identical Glu(551) to Arg(604) peptide from a neighbori ng Ca2+-ATPase polypeptide chain.