Hunter I, Schulthess T, Bruch M, Beck K, Engel J
Abteilung Biophysikalische Chemie, Biozentrum der Universität, Basel, Switzerland.
Eur J Biochem. 1990 Mar 10;188(2):205-11. doi: 10.1111/j.1432-1033.1990.tb15391.x.
The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.
利用源自该分子长臂的片段E8和C8-9研究层粘连蛋白链组装的特异性，其杆状结构域由A、B1和B2链的α-螺旋区域组成。通过横向尿素/聚丙烯酰胺凝胶电泳（PAGE）和圆二色性监测尿素诱导的链分离和展开。A链与二硫键连接的B1-B2链在3.5-4.0 M尿素时发生分离，到7.0 M尿素时所有残留的α-螺旋性丧失。通过透析去除尿素可使复性蛋白具有高回收率（87-100%），其表观分子量、α-螺旋含量、链组成、缔合程度和超微结构外观与天然E8无法区分。链的还原或还原并烷基化不会导致其特异性重新组装能力的降低。连接B1和B2链的单链间二硫键的重新形成清楚地表明，这些链在从其还原链复性的E8中正确地平行排列且对齐。从其还原并烷基化的链复性E8排除了二硫键形成在确定链排列中的作用，但表明它参与了正确组装分子的稳定化。这些结果与最近的序列数据一起，为A、B1和B2链的α-螺旋区域在分子长臂内形成三股卷曲螺旋中的相互作用提供了证据。这种相互作用的高度特异性表明它是层粘连蛋白在体内组装的机制。
