Markl J, Decker H, Linzen B, Schutter W G, von Bruggen E F
Hoppe Seylers Z Physiol Chem. 1982 Jan;363(1):73-87. doi: 10.1515/bchm2.1982.363.1.73.
The role of the seven different subunits in the quaternary structure of the 24-meric (37 S) hemocyanin of the tarantula, Eurypelma californicum, was studied by reassembly experiments. Individual subunits and combinations of 2, 3, 4, etc. different subunits were incubated in a total concentration of 1-2 mg/ml overnight in Tris buffer, pH 7.5, omitting divalent cations. The reassembly mixtures were then analyzed by thinlayer gel filtration, electron microscopy, analytical ultracentrifugation, and polyacrylamide gel electrophoresis. At least at the low protein concentration employed, none of the isolated monomeric subunits (a, d, e, f, g) is capable of hexamer are likewise ineffective. The minimal number of monomers required for hexamer formation is three, but only about half of the 10 possible combinations was effective. The best results were obtained with a + f + g. Four and five monomers always yielded hexamers, although the composition of the latter is not known. The heterodimer bc is a prerequisite to go beyond the hexameric state. bc alone forms tetramers and large, strand-like aggregates. a shows a high affinity towards bc, trimers and tetramers being formed. Out of all combinations of bc and two monomers, only one (bc + a + g) produced hexamers or heptamers. With three monomers plus bc, dodecamers and larger structures (but no 24-mers) were obtained, but only if also a was present (bc + a + f + g being the best combination). If all subunits minus one monomer were combined, the hexamer/heptamer level was not exceeded if a was omitted. 24-mers were formed in appreciable yield only in one case, namely if e was absent. However, this reassembly product turned out to be unstable. To obtain stable 24-meric hemocyanin, the complete set of subunits had to be present. The roles of the different subunits are defined as follows: bc is a "core" subunit which forms the inter-hexamer link within each dodecameric half. It serves also in inter-dodecamer bonding. The artificial homodimers bb and cc are ineffective in this respect. bc must be supplemented by a which is needed for dodecamer stabilization. f is also required to link two dodecamers, while g serves to stabilize this bridge. d and e are required to finish off and to stabilize the 24-mer. A model is proposed showing some neighbourhood relationships of the subunits within the hemocyanin 24-mer, and a pathway of reassembly is discussed.
通过重组实验研究了七种不同亚基在捕鸟蛛（Eurypelma californicum）24聚体（37S）血蓝蛋白四级结构中的作用。将单个亚基以及2、3、4等不同亚基的组合在Tris缓冲液（pH 7.5）中，以1 - 2 mg/ml的总浓度孵育过夜，省去二价阳离子。然后通过薄层层析凝胶过滤、电子显微镜、分析超速离心和聚丙烯酰胺凝胶电泳对重组混合物进行分析。至少在所采用的低蛋白浓度下，分离出的单体亚基（a、d、e、f、g）均不能形成六聚体，同样地，二聚体也是无效的。形成六聚体所需的单体最小数量是三个，但10种可能的组合中只有约一半是有效的。a + f + g组合得到的结果最佳。四个和五个单体总能形成六聚体，不过后者的组成尚不清楚。异二聚体bc是超越六聚体状态的前提条件。单独的bc形成四聚体和大的链状聚集体。a对bc具有高亲和力，能形成三聚体和四聚体。在bc与两个单体的所有组合中，只有一种（bc + a + g）产生六聚体或七聚体。三个单体加bc能得到十二聚体和更大的结构（但没有24聚体），但前提是也存在a（bc + a + f + g是最佳组合）。如果将所有亚基减去一个单体进行组合，如果省略a，则不会超过六聚体/七聚体水平。只有在一种情况下能以可观的产率形成24聚体，即不存在e时。然而，这种重组产物被证明是不稳定的。为了获得稳定的24聚体血蓝蛋白，必须存在全套亚基。不同亚基的作用定义如下：bc是一个“核心”亚基，在每个十二聚体半分子内形成六聚体间的连接。它也用于十二聚体间的结合。人工同二聚体bb和cc在这方面是无效的。bc必须由a补充，a是稳定十二聚体所必需的。f也是连接两个十二聚体所必需的，而g用于稳定这个桥。d和e是完成并稳定24聚体所必需的。提出了一个模型，展示了血蓝蛋白24聚体内亚基的一些相邻关系，并讨论了重组途径。
