Characterization of a 48‐kDa nucleic‐acid‐binding fragment of nucleolin

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Abstract
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.