Harris J R, Scheffler D, Gebauer W, Lehnert R, Markl J
Institute of Zoology, University of Mainz, Germany.
Micron. 2000 Dec;31(6):613-22. doi: 10.1016/s0968-4328(99)00145-6.
The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.
通过对负染标本进行透射电子显微镜（TEM）观察，评估了皱纹盘鲍血蓝蛋白两种同工型（HtH1和HtH2）的多聚体/更高寡聚体状态，以便与先前发表的钥孔戚血蓝蛋白（KLH1和KLH2）的结构数据进行比较[见Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. 钥孔戚血蓝蛋白（KLH），I：从Immucothel重聚，随后分离KLH1和KLH2。《微米》，28，31 - 41；Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. 钥孔戚血蓝蛋白（KLH）。II：纯化的KLH1和KLH2的特征性重聚特性。《微米》，28，43 - 56；Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. 钥孔戚血蓝蛋白（KLH）：KLH1和KLH2从Immucothel的体外缓慢重聚。《微米》，29，329 - 339]。在两种HtH同工型的纯化样品中，空心圆柱形约8MDa的十二聚体占主导，同时还有少量十聚体，但三聚体和更长的多聚体仅在HtH2中可检测到。监测了两种HtH同工型在不同离子条件下 的稳定性，从而确定了产生稳定十聚体的条件。评估了这些十聚体在Tris - HCl缓冲液（pH 7.4）中存在10和100mM浓度的钙和镁离子时重新形成多聚体的能力，以及单个HtH1和HtH2亚基（通过在甘氨酸 - NaOH缓冲液中pH 9.6解离产生）在钙和镁离子存在下重新缔合的能力。对于HtH1十聚体，在10和10mM钙和镁浓度下，主要的多聚体产物是十二聚体，而对于HtH2十聚体，则产生大量的多聚体，在较高的钙和镁浓度下反应进行得更完全。对于HtH1亚基，在10和100mM钙和镁离子存在下重新缔合，几乎100%转化为十二聚体，而HtH2亚基产生的混合物包含大量短的多聚体和相对较少的十二聚体，以及相当数量的较小直径的螺旋/管状聚合物。HtH1和HtH2十聚体的缔合特性以及亚基重新缔合，有力地表明了在所使用的实验条件下蛋白质的完整性和结构活性。还给出了KLH2十聚体缔合的数据，连同先前发表的关于KLH1十聚体缔合以及KLH1和KLH2亚基重新缔合的数据，使得能够对这两种软体动物的血蓝蛋白同工型进行详细比较。通过对负染TEM标本的研究，可以有效地评估软体动物血蓝蛋白寡聚体状态和亚基重新缔合的生化操作。
