Purification and characterization of an early protein (E14K) from adenovirus type 2-infected cells.
One adenovirus type 2 (Ad2) early protein, with an apparent molecular weight of 14,000 in sodium dodecyl sulfate-polyacrylamide gels (E14K), was purified to homogeneity. Purification involved fractionation of cytoplasmic extracts, precipitation at low pH, and DEAE-cellulose, phosphocellulose, and hydroxylapatite chromatography. The yield was around 12 microgram of purified protein per 10(9) HeLa cells. The two Ad2 DNA binding proteins with molecular weights of 75,000 and 45,000 (E75K and E45K) were purified by the same procedure. Tryptic peptide analyses indicated that the E14K protein is unrelated to the DNA binding proteins. The purified E14K protein has a high content of basic amino acids and a sedimentation coefficient of 5.5S in the native state, corresponding to a molecular weight of around 95,000. Pulse-chase experiments suggest that the E14K polypeptide is a primary translation product. Immunoprecipitation with a monospecific antiserum against the E14K protein revealed that it is exclusively localized in the cytoplasm of infected cells. E14K started to be synthesized at 2 hpostinfection, with a maximal rate of synthesis at 4 to 6 h postinfection. Immunoprecipitation of cell extracts from four different Ad2-transformed hamster embryo cell lines revealed that only one (Ad2HE4) of them expresses this protein. The adenovirus-simian virus 40 hybrid virus (Ad2ND1) does not express this protein, suggesting that the gene for the E14K protein is located in the part of the Ad2 genome which is deleted in this hybrid virus.
D000260:Adenoviruses, Human / Q000032:analysis* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D006367:HeLa Cells; D008970:Molecular Weight; D010452:Peptide Biosynthesis; D010455:Peptides / Q000032:analysis; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification
D000596:Amino Acids; D010455:Peptides; D014764:Viral Proteins