Isolation and characterization of a 125-kilodalton rapidly labeled nucleolar phosphoprotein.
A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.
D000818:Animals; D002466:Cell Nucleolus / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008114:Liver Neoplasms, Experimental / Q000032:analysis*; D008970:Molecular Weight; D009841:Oligonucleotides; D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000302:isolation & purification*; D010761:Phosphorus Radioisotopes; D011485:Protein Binding; D051381:Rats; D014357:Trypsin
D009841:Oligonucleotides; D010446:Peptide Fragments; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D014357:Trypsin