Third component of human complement: localization of the internal thiolester bond.
Third component of human complement: localization of the internal thiolester bond.
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人类补体的第三个组成部分：内部硫醇酯键的定位。
DOI：
10.1073/pnas.79.4.1054
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发表时间：
1982
影响因子：
11.1
通讯作者：
Tack,BF
中科院分区：
文献类型：
--
作者：
Thomas,ML;Janatova,J;Gray,WR;Tack,BF
人补体蛋白C3通过使用甲胺失活，从而从内部硫醇酯产生SH基团。通过该SH基团将蛋白偶联至活化的巯基-琼脂糖并用弹性蛋白酶消化。片段C3d保持与巯基-琼脂糖凝胶连接，随后用L-半胱氨酸洗脱。同时，原始SH基团再生，然后用碘[2 - 3H]乙酸标记。放射性标记的C3d片段的部分序列分析表明，硫羟酸酯的两种组分均位于氨基末端附近（残基23和26）。巯基经S-氰基化后，α链发生特异性化学断裂。获得的两个片段对应于氨基末端部分（Mr = 46，000）和羧基末端部分（Mr = 70，000）。这些结果共同表明片段C3d占据α链的大约345 - 610位。通过完成先前描述的胰蛋白酶肽的序列来延伸C3d的部分序列。C3 d的残基1 - 49与来自α 2-巨球蛋白的肽的比较[Swenson，R. P.&霍华德，J. B.（1980）J.Biol.Chem.255，8087 - 8091]显示了先前认可的硫羟酸酯位点周围的7个残基的同一性和α 2-巨球蛋白的已知糖基化位点周围的第二同一性区域。讨论了这些蛋白与C4蛋白的关系。C3的结构的总体轮廓，显示各种片段和切割位点的位置。硫醇酯基团的地方限制的局部折叠的肽链，一个可能的构象的建议和讨论有关的激活机制。
Human complement protein C3 was inactivated by using methylamine and thereby generating a SH group from the internal thiol ester. The protein was coupled via this SH group to activated thiol-Sepharose and digested with elastase. Fragment C3d remained attached to the thiol-Sepharose and was subsequently eluted with L-cysteine. Concomitantly, the original SH group was regenerated, and it was then labeled with iodo[2-3H]acetic acid. Partial sequence analysis of the radiolabeled C3d fragment showed that both components of the thiol ester are located close to the amino terminus (residues 23 and 26). Specific chemical cleavage of the α-chain was achieved afterS-cyanylation of the thiol. The two fragments obtained corresponded to the amino-terminal section (Mr≈46,000) and the carboxy-terminal section (Mr≈70,000). These results together indicate that fragment C3d occupies approximately positions 345-610 of the α-chain. The partial sequence of C3d was extended by completion of the sequence of a previously described tryptic peptide. Comparison of residues 1-49 of C3d with a peptide from α2-macroglobulin [Swenson, R. P. & Howard, J. B. (1980)J. Biol. Chem.255, 8087-8091] shows a previously recognized identity of seven residues around the thiol ester site and a second region of identity around a known glycosylation site of α2-macroglobulin. The relationships among these proteins and protein C4 are discussed. An overall outline of the structure of C3 is presented, showing the locations of various fragments and cleavage sites. The thiol ester group places constraints on the local folding of the peptide chain; a possible conformation is suggested and discussed in relation to the mechanism of activation.