Synthesis of vaccinia viral proteins in cytoplasmic extracts. I. Incorporation of radioactively labeled amino acids into polypeptides.
Polypeptide synthesis has been studied in cell-free systems prepared from vaccinia virus-infected and uninfected HeLa cells. Cytoplasmic extracts containing endogenous messenger ribonucleic acid were used. Amino acid incorporation into hot trichloroacetic acid-precipitable material was linear for 15 to 20 min at 37 C. The initial rate of protein synthesis was approximately 15% of the rate in intact cells. Optimal conditions for polypeptide synthesis were similar in cell-free systems prepared from infected or uninfected cells. Requirements for an energy source and Mg(++) were demonstrated. The optimal Mg(++) concentration was 4 to 5 mm. Ribonuclease, puromycin, and cycloheximide were inhibitory. The molecular weights of the polypeptides labeled in the cell-free systems, as determined by gel filtration in 5 m guanidine hydrochloride, ranged from 16,000 to above 68,000. Polyacrylamide gel electrophoresis indicated that the polypeptides labeled in cell-free extracts of uninfected and infected cells were different. The latter closely corresponded in electrophoretic mobility with the viral polypeptides made in intact, infected cells.
D000596:Amino Acids / Q000378:metabolism; D001345:Autoradiography; D002247:Carbon Isotopes; D002474:Cell-Free System; D002850:Chromatography, Gel; D003513:Cycloheximide / Q000494:pharmacology; D003851:Deoxyribonucleases / Q000494:pharmacology; D004586:Electrophoresis; D006367:HeLa Cells; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D010452:Peptide Biosynthesis; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology; D012260:Ribonucleases / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014616:Vaccinia virus / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*
D000596:Amino Acids; D002247:Carbon Isotopes; D010455:Peptides; D014764:Viral Proteins; D010649:Phenylalanine; D011691:Puromycin; D003513:Cycloheximide; D003851:Deoxyribonucleases; D012260:Ribonucleases; D008274:Magnesium
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