Lueders K K, Kuff E L
J Biol Chem. 1975 Jul 10;250(13):5192-9.
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.
在组织培养的小鼠神经母细胞瘤细胞中研究了脑池内A颗粒主要结构蛋白（P73）的合成与周转。制备了含有细胞中95% A颗粒抗原的Triton X - 100:EDTA不溶性沉淀，并通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳进行分析。在含有大量A颗粒的三株神经母细胞瘤细胞系的沉淀中，一种分子量为73,000的成分很突出，该成分被鉴定为A颗粒结构蛋白P73。在不含A颗粒的细胞制备的沉淀中则不存在该成分。标记氨基酸掺入P73的量约占细胞总掺入量的1.2%，当细胞生长从对数期转变为稳定期时，这一比例没有变化。放射性氨基酸添加到培养基后2分钟内，标记就出现在P73上。脉冲追踪和抑制剂研究在证明未组装成A颗粒的P73池很小时，证实了抗原测量结果。周转研究表明，P73获得和失去标记的速度比平均细胞蛋白更快。在一个经过充分表征的细胞系中，估计约60%的主要A颗粒蛋白在24小时内周转。尽管细胞将合成的约10%的蛋白质释放到培养液中，但A颗粒蛋白似乎没有释放。对培养液的分析未发现A颗粒、可溶性A颗粒蛋白或A颗粒抗原。因此，似乎这些颗粒合成和降解相对较快，并且周转完全发生在细胞内。
