Lamy J, You V, Taveau J C, Boisset N, Lamy J N
Laboratoire des Protéines Complexes, Campus Médecine, 2 bis Boulevard Tonnellé, Tours Cedex, F-37032, France.
J Mol Biol. 1998 Dec 11;284(4):1051-74. doi: 10.1006/jmbi.1998.2235.
The quaternary structure of Sepia officinalis hemocyanin (Hc) as studied in immunoelectron microscopy with rabbit IgGs and Fab fragments raised against functional units (FU) Soc, Sod, Soe, Sof, Sog, and Soh and fragment Soab. The architecture of immunocomplexes shows that (i) epitopes characteristic of FUs Soc and Sog and of fragment Soab are located in the two external tiers of FUs, (ii) FUs Soh and Soe or Sod are located in arches. These results were confirmed using immunocomplexes made up of Sepia Hc and IgGs or Fab fragments purified from antisera raised against FUs of Octopus vulgaris and Octopus dofleini. Frozen-hydrated immunocomplexes containing one Hc molecule and at least one FU-specific Fab fragment were observed in the electron microscope and submitted to image processing. When the Hc molecule is viewed along its 5-fold axis (i) anti-Soc Fab fragments project on a radius passing through the arch's pillar, (ii) anti-Sof Fabs project slightly out of the arches, and (iii) anti-Soh Fabs project between neighboring arches. When applied to a recent three-dimensional (3D) reconstruction volume, these results allow us to deduce the intramolecular location of five of the eight FUs. For the last three FUs limited uncertainties remain: (i) Soc can be located in two positions in the external tier of FUs; (ii) Soa and Sob can both occupy three positions in the external tiers; and (iii) because of an immunological cross-reactivity Sod may be located in the wall and Soe in the arch, or vice versa. An analysis of the quaternary structure considering the possible locations of the 80 FUs and postulating a single type of subunit shows that 80 possibilities of paths still exist for the polypeptide chain. To solve definitely these 80 possibilities only five questions remain to be answered.
利用针对功能单元（FU）Soc、Sod、Soe、Sof、Sog和Soh以及片段Soab产生的兔IgG和Fab片段，通过免疫电子显微镜研究了乌贼血蓝蛋白（Hc）的四级结构。免疫复合物的结构表明：（i）FU Soc和Sog以及片段Soab的特征性表位位于FU的两个外部层级；（ii）FU Soh和Soe或Sod位于拱中。使用由乌贼Hc与从针对普通章鱼和多氏章鱼的FU产生的抗血清中纯化的IgG或Fab片段组成的免疫复合物，证实了这些结果。在电子显微镜下观察含有一个Hc分子和至少一个FU特异性Fab片段的冷冻水合免疫复合物，并进行图像处理。当沿Hc分子的5重轴观察时：（i）抗Soc Fab片段投射在穿过拱支柱的半径上；（ii）抗Sof Fab片段略微伸出拱外；（iii）抗Soh Fab片段投射在相邻拱之间。将这些结果应用于最近的三维（3D）重建体积时，我们可以推断出八个FU中五个的分子内位置。对于最后三个FU，仍存在有限的不确定性：（i）Soc可位于FU外部层级的两个位置；（ii）Soa和Sob均可在外部层级占据三个位置；（iii）由于免疫交叉反应，Sod可能位于壁中，而Soe位于拱中，反之亦然。考虑到80个FU的可能位置并假设单一类型的亚基对四级结构进行分析表明，多肽链仍存在80种可能的路径。要明确解决这80种可能性，只剩下五个问题有待回答。
