Purification and characterization of a platelet-derived growth factor and heavy metal-modulated nuclear protein.
Platelet-derived growth factor (PDGF), fibroblast growth factor, and heavy metal salts such as sodium arsenite stimulated BALB/c-3T3 cells to synthesize a 31-kDa protein(s) (termed p31) in a concentration-dependent manner. p31 was also synthesized in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). V8 protease digestion of p31 purified from PDGF-, TPA-, and arsenite-treated cells showed identical fragmentation patterns, demonstrating that these agents modulate synthesis of the same (or a highly similar) protein. TPA-induced p31 synthesis was cell cycle-specific, occurring in density-arrested but not exponentially replicating cells. p31 was readily labeled with [35S]methionine but not with [35S]cysteine. Thus it is not a metallothionein. The protein associated with nuclei. It appears to be highly hydrophobic because solubilization required detergents or organic solvents. Reverse-phase high performance liquid chromatography (HPLC) provided further evidence of hydrophobicity. p31 has been purified to homogeneity using sodium dodecyl sulfate gels with electroelution and reverse-phase HPLC. It has an isoelectric point of 6.8. Its nuclear localization and amino acid analysis demonstrate that p31 is not heme oxygenase, a 32-kDa arsenite-induced microsomal protein. Stimulation of p31 synthesis by growth factors, PDGF and fibroblast growth factor; a tumor promoter, TPA; and heavy metal salts suggests that there is overlap in the pathways for mitogenic stimulation and heavy metal stress.
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Department of Pediatrics, Children's Hospital of Philadelphia, Pennsylvania 19104.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001151:Arsenic / Q000494:pharmacology*; D018053:Arsenites; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000302:isolation & purification; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017670:Sodium Compounds; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000596:Amino Acids; D018053:Arsenites; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D017670:Sodium Compounds; C017947:sodium arsenite; D001151:Arsenic; D013755:Tetradecanoylphorbol Acetate