Reconstitution of the filamentous backbone of lens beaded-chain filaments from a purified 49kD polypeptide.
The beaded-chain filaments unique to the fiber cells of the crystalline lens are composed of a linear array of spheroidal particles which appear to be connected by a filamentous backbone. In order to determine the existence of the putative backbone and to characterize its constituents, one of the major proteins associated with beaded-chains in the chicken lens was investigated. 49kD was isolated in an enriched fraction derived from the 8M urea extract of the lens cell water-insoluble residue. The polypeptide (which exists in several charge isoforms, the major at pI 5.2) was purified sequentially by gel filtration on Sephacryl S-200, hydrophobic interaction chromatography on phenyl-Sepharose, and anionic exchange chromatography on Mono Q, all under denaturing conditions. Immunoblot analyses established that 49kD was immunologically distinct from vimentin, actin, and tubulin/MAPs (representing the three classes of cytoplasmic filaments), as well as from the crystallins. Amino acid analyses demonstrated compositional differences for 49kD compared with lens actin and vimentin, and one- and two-dimensional peptide mapping of 49kD and vimentin revealed no homology. Electron microscopy demonstrated that short, contorted filaments were produced upon removal of purified 49kD from urea to low-salt buffers. In the presence of physiological salt concentrations 49kD assembled into extensive 4-6nm diameter, straight filaments similar to the backbone seen in native beaded-chain filaments, but morphologically distinct from the other cytoplasmic filament classes.
Lieska
N
N
Department of Anatomy, University of Illinois College of Medicine, Chicago 60612.
Yang
H Y
HY
Maisel
H
H
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007526:Isoelectric Point; D007908:Lens, Crystalline / Q000737:chemistry*
D000596:Amino Acids; D003598:Cytoskeletal Proteins