Identification of the disulfide-linked peptide in irreversibly sickled cell beta-actin
Identification of the disulfide-linked peptide in irreversibly sickled cell beta-actin
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DOI：
10.1021/bi960063n
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发表时间：
1996-04-09
期刊：
影响因子：
2.9
通讯作者：
Goodman, SR
中科院分区：
文献类型：
--
作者：
Bencsath, FA;Shartava, A;Goodman, SR
我们之前已经证明，在高离子强度的Triton X-100缓冲液中，不可逆镰状细胞（ISCs）的膜骨架在37℃时解离的速度比可逆镰状细胞或对照红细胞的膜骨架要慢[Shartava et al. (1995) J. Cell.]。此外，我们证明了这种“缓慢解离”的主要原因是ISC β -actin的一个翻译后修饰。由于这种修饰，两个巯基（Cys（284）和Cys(373)）变得不能被硫醇试剂获得。由于还原剂二硫苏糖醇还原了巯基，因此我们认为该修饰可能是Cys（284）和Cys（373）之间的二硫桥接。在这篇文章中，我们直接证明了半胱氨酸（284）和半胱氨酸（373）在ISC β -actin中存在二硫桥。我们合成了相关的ISC β -actin - tryptic胱氨酸肽（KCF-CDVDIR），通过HPLC、MS和MSMS对其进行了表征，并在ISC β -actin - tryptic digest中进行了鉴定。这些结果支持了我们之前的建议，即ISC β -肌动蛋白的氧化变化是不可逆镰状细胞现象的主要原因。
We have previously demonstrated that the membrane skeletons of irreversibly sickled cells (ISCs) dissociate more slowly at 37 degrees C, in high ionic strength Triton X-100 buffer, than do the membrane skeletons of reversibly sickled cells or control erythrocytes [Shartava et al. (1995) J. Cell. Biol. 128, 805-818], Furthermore, we demonstrated that the major cause of this' slow dissociation was a single posttranslational modification in ISC beta-actin. Two sulfhydryl groups (Cys(284) and Cys(373)) became inaccessible to thiol reagents because of this modification. We suggested the possibility that the modification was a disulfide bridge between Cys(284) and Cys(373) since the reducing agent dithiothreitol restored the sulfhydryl groups. In this article, we directly demonstrate the existence of the disulfide bridge between cysteine(284) and cysteine(373) in ISC beta-actin. We synthesized the associated ISC beta-actin tryptic cystine-peptide (KCF-CDVDIR), characterized it by HPLC, MS, and MSMS, and identified it in the tryptic digest of the ISC beta-actin. These results support our earlier suggestion that the oxidative change in ISC beta-actin is a major cause of the irreversible sickling phenomenon.