Felipo V
Physiol Chem Phys Med NMR. 1986;18(1):25-32.
A rat liver cytosol was used to study protein synthesis per se and also to study import of proteins into mitochondria since rat liver cytosol represents an environment closer to that of liver mitochondria than the generally used reticulocytes lysates. Two ATP-regenerating systems were compared. The creatine phosphate/creatine kinase yields higher protein synthesis than the phosphoenol pyruvate/pyruvate kinase system. Hemin, necessary to maintain synthesis by reticulocyte lysates, does not affect the rat liver cytosol. The level of protein synthesis obtained with this cell-free system is comparable to other eukaryotic systems described recently and to the expected value for "in vivo" conditions. Isolated mitochondria incorporated, under our standard conditions, newly synthesized proteins linearly up to 30 min, it ceases when a component(s) in the cytosol had been depleted; addition of freshly translated cytosol restores the import. The bulk of imported proteins are retained in mitoplasts or in mitochondria after treatment with trypsin. The cytosol system will be useful to study questions such as regulation of liver mRNA translation and mitochondrial protein turnover.
大鼠肝细胞溶胶被用于研究蛋白质合成本身，也用于研究蛋白质向线粒体的导入，因为与常用的网织红细胞裂解物相比，大鼠肝细胞溶胶代表了一种更接近肝线粒体的环境。对两种ATP再生系统进行了比较。磷酸肌酸/肌酸激酶产生的蛋白质合成比磷酸烯醇丙酮酸/丙酮酸激酶系统更高。维持网织红细胞裂解物合成所必需的血红素，对大鼠肝细胞溶胶没有影响。用这种无细胞系统获得的蛋白质合成水平与最近描述的其他真核系统以及“体内”条件下的预期值相当。在我们的标准条件下，分离的线粒体可线性地整合新合成的蛋白质长达30分钟，当细胞溶胶中的一种或多种成分耗尽时，整合停止；添加新翻译的细胞溶胶可恢复导入。在用胰蛋白酶处理后，大部分导入的蛋白质保留在线粒体膜间隙或线粒体中。该细胞溶胶系统将有助于研究诸如肝mRNA翻译的调节和线粒体蛋白质周转等问题。
