Steinert P M
Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 May 25;265(15):8766-74.
The composition of the two-chain coiled-coil molecule of murine epidermal keratin intermediate filaments (KIF) containing keratins 1 (type II) and 10 (type I) has been explored using native-type KIF as well as KIF reassembled in vitro from protein dissolved in urea solutions or from mixtures of 3H-labeled and unlabeled purified chains. By use of cross-linking, high resolution polyacrylamide gel electrophoresis and blotting for 3H-labeled keratins or with an anti-mouse keratin 10 antibody, it was found that individual keratin chains form type I or type II homodimers and homotetramers in solution that do not assemble into KIF in vitro. When mixed in urea solutions of 5 M or greater, such homo-oligomers rapidly rearrange into mostly heterodimers and heterotetramers that support filament assembly. On the other hand, prekeratin, isolated from newborn mouse epidermis with 0.1 M sodium citrate buffer, pH 2.6, under conditions that do not dissociate the native coiled-coil molecule, consists exclusively of type I-II heterodimers and heterotetramers. It is necessary to dissolve prekeratin in 8-9.5 M urea for several hours in order to dissociate the native heterodimer molecule and incorporate tracer amounts of a single 3H-labeled keratin chain. These data establish that native KIF consist of heterodimer coiled-coil molecules. Furthermore, heterodimers are much more stable than homodimers and are the favored form of association in solution. However, some homodimers (10-30% of total) always form after dissolution in concentrated urea and can be assimilated into KIF during reassembly in vitro. The isolation of alpha-helix-enriched dimer particles from the 2B region of the rod domains upon limited proteolysis confirmed the presence of mostly heterodimer and some homodimer molecules in reassembled KIF. These properties of keratin chains in urea solutions hereby clarify a number of conflicting reports in the literature concerning the composition of the coiled-coil molecule. The presence of some homo-oligomeric species in reassembled KIF correlates with earlier observations of polymorphism as well as stoichiometry.
利用天然型小鼠表皮角蛋白中间丝（KIF）以及从溶解于尿素溶液中的蛋白质或从³H标记和未标记的纯化链混合物体外重组装的KIF，对包含角蛋白1（II型）和角蛋白10（I型）的双链卷曲螺旋分子的组成进行了研究。通过交联、高分辨率聚丙烯酰胺凝胶电泳以及对³H标记角蛋白或用抗小鼠角蛋白10抗体进行印迹分析，发现单个角蛋白链在溶液中形成I型或II型同二聚体和同四聚体，这些在体外不会组装成KIF。当在5M或更高浓度的尿素溶液中混合时，此类同寡聚体迅速重排为大多是异二聚体和异四聚体，它们支持丝状体组装。另一方面，在不会使天然卷曲螺旋分子解离的条件下，用0.1M柠檬酸钠缓冲液（pH2.6）从新生小鼠表皮分离的前角蛋白仅由I-II型异二聚体和异四聚体组成。为了解离天然异二聚体分子并掺入微量的单条³H标记角蛋白链，有必要将前角蛋白溶解于8 - 9.5M尿素中数小时。这些数据表明天然KIF由异二聚体卷曲螺旋分子组成。此外，异二聚体比同二聚体稳定得多，并且是溶液中更倾向的缔合形式。然而，在溶解于浓尿素后总会形成一些同二聚体（占总量的10 - 30%），并且在体外重组装过程中可被纳入KIF。通过有限蛋白酶解从杆状结构域的2B区域分离富含α螺旋的二聚体颗粒，证实了重组装的KIF中大多是异二聚体分子以及一些同二聚体分子的存在。尿素溶液中角蛋白链的这些特性从而澄清了文献中关于卷曲螺旋分子组成的一些相互矛盾的报道。重组装的KIF中一些同寡聚体物种的存在与早期关于多态性以及化学计量学的观察结果相关。
