Biochemical characterization of mouse brain necdin.
Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on microsomal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits of pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (>200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.
Maruyama
E
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Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Japan.
D000375:Aging; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D001923:Brain Chemistry; D018236:Carcinoma, Embryonal; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009707:Nucleosomes / Q000737:chemistry; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; C069766:necdin