Plectin from bovine lenses. Chemical properties, structural analysis and initial identification of interaction partners.
Plectin was purified to near homogeneity from epithelial and cortical cell layers of bovine lenses using a simple and fast purification scheme that included as last step, gel permeation chromatography in the presence of 0.25% sodium N-lauroyl sarcosinate. Lens cell plectin showed extensive structural homology to plectin from cultured cells as revealed by immunoblotting experiments and amino acid analysis. Further characterization included solubility in various buffer solutions, codistribution with vimentin in repeated rounds of intermediate filament disassembly and assembly, and hydrodynamic behaviour in high-performance gel permeation chromatography. Electron microscopy of negatively stained and rotary shadowed plectin molecules revealed a dumb-bell-like structure with an estimated relative molecular mass of 1.16 X 10(6). Specific head-to-head self-interaction of plectin molecules at low salt concentrations and formation of large aggregates under high-salt and physiological conditions was also demonstrated. Isolation, as well as reconstitution of soluble protein complexes containing plectin, vimentin and other cytoskeletal and membrane skeleton proteins, provided first hints to plectin's role as an interlinking component of the cytoskeleton and the membrane skeleton of lens tissue.
Weitzer
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Institute of Biochemistry, University of Vienna, Austria.
Wiche
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003459:Crystallins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D007381:Intermediate Filament Proteins; D007908:Lens, Crystalline / Q000032:analysis*; D008854:Microscopy, Electron; D008970:Molecular Weight; D051190:Plectin; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D012995:Solubility; D014746:Vimentin / Q000032:analysis / Q000378:metabolism
D000596:Amino Acids; D003459:Crystallins; D007381:Intermediate Filament Proteins; D051190:Plectin; D011506:Proteins; D014746:Vimentin