Purification and characterization of a nuclear pore glycoprotein complex containing p62.
It is known that nucleoporins, a family of glycoproteins with N-acetylglucosamine that are found in nuclear pore complexes, are essential for nuclear import and export. A major component of the family, p62, was purified from a salt extract of rat liver nuclear envelopes by wheat germ agglutinin-Sepharose affinity chromatography and DEAE-anion exchange HPLC. p62 was purified as a complex with two glycoproteins of 60 and 54 kDa. The presence of the complex was confirmed by gel filtration, glycerol density gradient centrifugation, and cross-linking experiments. The molecular ratio of the 62-, 60-, and 54-kDa components of the complex was estimated to be 1: 1.1 +/- 0.2: 1.7 +/- 0.3 from the intensity of Coomassie Blue staining of SDS-PAGE gels. The complex was stable against 1 M NaCl, 1% Triton X-100, and 2 M urea. The Stokes' radius and sedimentation coefficient of the complex are 8.0 nm and 6.7 S. The molecular mass and frictional ratio of the complex were estimated to be about 231 kDa and 2.0, respectively. p62 and p54 were acidic and neutral proteins, respectively, exhibiting charge heterogeneities, and p60 was assumed to be a basic protein. p60 tended to undergo proteolytic degradation to a 47-kDa fragment.
Kita
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K
Department of Biochemistry, Faculty of Science, Niigata University.
Omata
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S
Horigome
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T
D000117:Acetylglucosamine / Q000737:chemistry; D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000737:chemistry*; D028861:Nuclear Pore Complex Proteins; D051381:Rats
D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; C049312:nuclear pore protein p62; D000117:Acetylglucosamine