Synthesis and turnover of intracisternal A-particle structural protein in cultured neuroblastoma cells.
Synthesis and turnover of the main structural protein (P73) of intracisternal A-particles were studied in mouse neuroblastoma cells in tissue culture. Triton X-100:EDTA-insoluble pellets containing 95% of the A-particle antigen in the cells were prepared and analyzed by electrophoresis in Na dodecyl-SO4-minus polyacrylamide gels. A 73,000 molecular weight component was prominent in pellets from three lines of neuroblastoma which contain numerous A-particles and this component was identified as the A-particle structural protein P73. It was absent in pellets prepared from cells which do not contain A-particles. Incorporation of labeled amino acids into P73 represented approximately 1.2% of total cell incorporation and this proportion did not change when the cell growth changed from log phase to stationary phase. Label appeared P73 within 2 min after radioactive amino acids were added to the medium. Pulse-chase and inhibitor studies confirmed antigenic measurements in demonstrating that the pool of P73 not assembled into A-particles was small. Turnover studies showed that P73 gained and lost label more rapidly than the average cell protein. In one cell line which was thoroughly characterized, approximately 60% of the main A-particle protein was estimated to turn over in a 24-hour period. Although the cells released approximately 10% of the proteins synthesized into the culture fluid, A-particle protein did not appear to be released. Analysis of culture fluid failed to reveal A-particles, soluble A-particle proteins, or A-particle antigen. It appears, therefore, that the particles are relatively rapidly synthesized and degraded, and that turnover occurs entirely intracellularly.
D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D003168:Complement Fixation Tests; D004591:Electrophoresis, Polyacrylamide Gel; D007930:Leucine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000378:metabolism; D009374:Neoplasms, Experimental; D009447:Neuroblastoma / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D012513:Sarcoma, Experimental; D013539:Simian virus 40; D012967:Sodium Dodecyl Sulfate; D014316:Tritium; D014633:Valine / Q000378:metabolism
D002250:Carbon Radioisotopes; D009363:Neoplasm Proteins; D014316:Tritium; D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine; D007930:Leucine; D014633:Valine; D008239:Lysine