Purification and characterization of a 65-kDa tumor-associated phosphoprotein from rat transplantable hepatocellular carcinoma 1682C cell line.
We have isolated a homogeneous tumor-associated phosphoglycoprotein of about 65 kDa (p65) by ammonium sulfate precipitation of proteins from conditioned medium containing the rat transplantable hepatocellular carcinoma 1682C cell line, followed by high-performance liquid chromatography on molecular-sieving and phenyl hydrophobic interaction columns. The protein was concentrated in a Rotofor isoelectric focusing cell and finally separated by isoelectrofocusing followed by SDS--polyacrylamide gel electrophoresis. We achieved a purification of approximately 11,000-fold after the Rotofor concentration step. This protein migrated as a single band upon electrophoresis in SDS-PAGE and had a pI of 5.8 in isoelectrofocusing gels. The carbohydrate content of the blotted phosphoglycoprotein was analyzed by probing the blots with biotinylated lectins; a positive reaction was detected with concanavalin A, wheat-germ agglutinine, and Ricinus communis agglutinine. To confirm the tumor origin of this molecule, hepatocellular carcinoma cells were labeled in vivo using [32P]orthophosphate as well as [35S]methionine and cell culture medium was analyzed for the presence of radioactive band that corresponds with our protein. Phosphoamine acid analysis by thin-layer chromatography showed the presence of phosphotyrosine, phosphothreonine, and phosphoserine, which was later confirmed by analysis of the amino acid composition. Using the method described by Marchalonis and Weltman for comparative analysis of protein structure and evolution, we compared the protein isolated by us with other tumor markers and proteins showing similar properties and found no significant similarities.
Mirowski
M
M
University of Texas M. D. Anderson Cancer Center, Smithville 78957.
Sherman
U
U
Hanausek
M
M
D000818:Animals; D014408:Biomarkers, Tumor / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008114:Liver Neoplasms, Experimental / Q000737:chemistry* / Q000473:pathology; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D014407:Tumor Cells, Cultured / Q000737:chemistry
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; C077457:tumor-associated phosphoprotein p65