We have examined the oxidative sensitivity of theCa
2+-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
M AAPH-derived radicals results in a concentration- and time-dependentinhibition of calcium-dependent ATPaseactivity correlating with the loss of monomericCa
2+-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 Ca
2+-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 theCa
2+-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 Ca
2+-ATPase:(i) Glu
121 to Lys
128, (ii) His
190to Lys
218, (iii) Asn
330 to Lys
352,(iv) Gly
432 to Lys
436, (v) Glu
551to Arg
604,and (vi) Glu
657 to Arg
671. TheGlu
551 to Arg
604 peptide, located within thenucleotide binding domain,was found to participate in the formation of intermolecular bityrosinecross-links with the identical Glu
551to Arg
604 peptide from a neighboringCa
2+-ATPase polypeptide chain.