Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase.
Identification of oxidation-sensitive peptides within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+-ATPase.
复制标题
DOI：
10.1021/bi970058z
复制
发表时间：
1997-06
期刊：
影响因子：
2.9
通讯作者：
Rosa I. Viner;A. Krainev;Todd A. Williams;Christian Schöneich;Diana J. Bigelow
中科院分区：
文献类型：
--
作者：
Rosa I. Viner;A. Krainev;Todd A. Williams;Christian Schöneich;Diana J. Bigelow
我们研究了骨骼肌肌浆网（SR）膜Ca ~（2+）-ATP酶的氧化敏感性，将分离的SR膜暴露于热不稳定的水溶性自由基引发剂2，2 ′-偶氮二（2-脒基丙烷）二盐酸盐（AAPH）。与高达702 μ M AAPH衍生的自由基孵育导致钙依赖性ATP酶活性的浓度和时间依赖性抑制，这与单体Ca 2 +-ATP酶多肽的损失以及伴随的较高分子量物质的出现有关。然而，没有检测到氧化剂诱导的蛋白质片段化。所观察到的氧化剂诱导的酪氨酸的形成占分子间Ca 2 +-ATP酶交联，以及分子内交联。在还原和非还原条件下进行SDS-PAGE检查后，排除了巯基氧化为二硫化物作为分子间交联的另一个可能来源。SR膜暴露于AAPH衍生的自由基物种导致小程度的脂质过氧化，这与酶失活无关，表明跨膜肽的修饰与酶失活无关。六个细胞质肽已被确定为通过暴露于AAPH或过氧化氢而被修饰，这表明Ca 2 +-ATP酶的这些区域通常对氧化剂敏感。在通过反相HPLC分离后，随后通过N-末端测序和氨基酸分析鉴定这些氧化肽，其对应于Ca 2 +-ATP酶的以下序列：（i）Glu 121至Lys 128，（ii）His 190至Lys 218，（iii）Asn 330至Lys 352，（iv）Gly 432至Lys 436，（v）Glu 551至Arg 604，和（vi）Glu 657至Arg 671。Glu 551至Arg 604肽，位于核苷酸结合结构域内，被发现参与与来自相邻Ca 2 +-ATP酶多肽链的相同Glu 551至Arg 604肽的分子间酪氨酸交联的形成。
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 microM 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.