Formation and identification of cytoskeletal components from liver cytosolic precursors.
Liver cytosol forms a macroscopic fibrillary network in the presence of low concentrations of MgCl2. This process represents the generation of 3- to 11-nm filaments from soluble precursors, involving selectively at least 12 major polypeptides. Similar polypeptides are enriched in the detergent-insoluble fraction from hepatocytes, suggesting that they may be important constituents of the native cytoskeleton. AcA 34 gel-permeation chromatography resolves the cytosol into three independently "polymerizing" peaks: A, B, and C. The formation of filaments follows biphasic kinetics in peaks B and C, whereas peak A lacks the slow phase. Filament formation in all three systems is inhibited by 1-15 mM inorganic phosphate, 10 mM NaF, or 10 mM sodium molybdate. The polymerization of peak C only is inhibited by 0.2-2 mM ATP. CaCl2 (1-100 microM) has no apparent regulatory effect. Two-dimensional polypeptide analysis and peptide mapping show that actin is a major component of peak C, while peaks A and B contain prominent polypeptides that may be related to intermediate filament subunits. In addition, all three systems contain two or three high molecular weight (greater than 170,000) polypeptides that may participate in modulating and extending the filament network. The filaments from peaks A and B are soluble in 8 M urea and reform on removal of the urea in the presence of 5 mM MgCl2. The polypeptide composition remains constant through three such cycles.
D000199:Actins / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D003600:Cytosol / Q000648:ultrastructure; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007526:Isoelectric Point; D007633:Keratins / Q000378:metabolism; D008099:Liver / Q000648:ultrastructure*; D008970:Molecular Weight; D011485:Protein Binding
D000199:Actins; D007381:Intermediate Filament Proteins; D007633:Keratins