BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF THE REOVIRUS CELL ATTACHMENT PROTEIN-SIGMA-1 - EVIDENCE THAT IT IS A HOMOTRIMER
BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF THE REOVIRUS CELL ATTACHMENT PROTEIN-SIGMA-1 - EVIDENCE THAT IT IS A HOMOTRIMER
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DOI：
10.1016/0042-6822(91)90818-v
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发表时间：
1991-09-01
期刊：
影响因子：
3.7
通讯作者：
LEE, PWK
中科院分区：
文献类型：
--
作者：
STRONG, JE;LEONE, G;LEE, PWK
通过生物化学和生物物理方法确定呼肠孤病毒细胞附着蛋白σ1（49 K单体分子量）的寡聚化状态。将体外合成的全长（蛋白产物指定为A）和C-末端截短的（蛋白产物指定为B）血清型3呼肠孤病毒S1 mRNA转录物在兔网织红细胞裂解物中共翻译，并在允许鉴定σ 1寡聚形式的条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析产物。一致地观察到总共四个寡聚蛋白条带（分别对应于A3、A2 B1、A1 B2和B3），这表明该蛋白由三个单体亚基组成。使用柱过滤和蔗糖梯度沉降分析纯化的σ1的生物物理表征证实了σ1的高度不对称形状，并允许我们确定天然蛋白质的分子量为132 K（三聚体）。对σ1的两个胰蛋白酶片段[N-末端纤维状尾部（26 K单体分子量）和C-末端球状头部（23 K单体分子量）]进行类似的生物物理分析，分别得出分子量为77 K和64 K，两者均再次对应于三聚体。因此，我们得出结论，蛋白质σ1是一个同源三聚体，并提供了支持性的实验证据，在SDS-聚丙烯酰胺凝胶中的异常行为的寡聚蛋白质，这，再加上化学交联的研究，在一定程度上导致了以前的建议，σ1可能是一个更高的顺序寡聚体的基本原理。
The oligomerization state of the reovirus cell attachment protein σ1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus Sl mRNA transcripts synthesizedin vitrowere cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of σl. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified σ1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of σ1 and allowed us to determine the molecular weight of the native protein to be ∼132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the σ1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein σ1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that σ1 might be a higher order oligomer.