Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point.
A monoclonal antibody was raised against a salt-extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an approximately 100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (pI) 5.5--6.1, characteristic for nucleolin, and at pI 8.5--9. Whereas the protein focusing at acidic pI was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic pI in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix-derived antigen was positioned at pI 5--6. The present data indicate that the nuclear matrix-bound nucleolin is associated with ribonucleoproteins and a basic component resisting dissociation under conditions of isoelectric focusing.
Gotzmann
J
J
Institute of Tumor Biology-Cancer Research, University of Vienna, Austria.
Eger
A
A
Meissner
M
M
Grimm
R
R
Gerner
C
C
Sauermann
G
G
Foisner
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D000941:Antigens / Q000032:analysis; D002460:Cell Line; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007526:Isoelectric Point; D015530:Nuclear Matrix / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis*; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000032:analysis*; D000097796:Nucleolin
D000911:Antibodies, Monoclonal; D000941:Antigens; D010750:Phosphoproteins; D012313:RNA; D016601:RNA-Binding Proteins; D000097796:Nucleolin