Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei.
The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D003840:Deoxycholic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000648:ultrastructure*; D008970:Molecular Weight; D009698:Nucleoproteins / Q000032:analysis
D009698:Nucleoproteins; D003840:Deoxycholic Acid