Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin.
Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.
Sapp
M
M
Fakultät für Biologie, Universität Konstanz.
Richter
A
A
Weisshart
K
K
Caizergues-Ferrer
M
M
Amalric
F
F
Wallace
M O
MO
Kirstein
M N
MN
Olson
M O
MO
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D002499:Centrifugation, Density Gradient; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D016601:RNA-Binding Proteins; D013950:Thymus Gland; D014357:Trypsin; D000097796:Nucleolin
D000596:Amino Acids; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D014357:Trypsin; D000097796:Nucleolin