Raymond Y, Shore G C
J Biol Chem. 1981 Mar 10;256(5):2087-90.
Pulse-chase experiments using liver explants incubated in modified Eagle's medium showed that newly synthesized precursor for carbamyl phosphate synthetase (pCPS) passes very rapidly through the cytosolic compartment en route to mitochondria (t 1/2 is approximately 2 min). Since even a small pool of precursor could not be detected in association with mitochondria, processing of pCPS must occur either coincident with or immediately following its transmembrane uptake by the organelle. Treatment of explants with the protease inhibitor, p-aminobenzamidine, however, inhibited normal processing of the precursor. But, rather than accumulate in the cell, newly synthesized pCPS was nonspecifically degraded with kinetics (t 1/2 of 2-3 min) which are consistent with the idea that the precursor was almost instantly degraded upon reaching the blocked processing enzyme in a mitochondrion. The protease inhibitor had little or no effect on synthesis of pCPS. This was determined by isolating polysomes aminobenzamidine; the two polysome preparations were about equally active in synthesizing pCPS in vitro in the presence of an inhibitor of polypeptide chain initiation, aurin tricarboxylic acid.
使用在改良伊格尔培养基中培养的肝外植体进行的脉冲追踪实验表明，新合成的氨甲酰磷酸合成酶（pCPS）前体在前往线粒体的途中非常迅速地穿过胞质区室（半衰期约为2分钟）。由于在与线粒体相关的区域甚至检测不到一小部分前体，因此pCPS的加工过程必定与该细胞器对其跨膜摄取同时发生或紧随其后。然而，用蛋白酶抑制剂对氨基苯甲脒处理外植体，会抑制前体的正常加工。但是，新合成的pCPS并非在细胞中积累，而是以动力学方式（半衰期为2 - 3分钟）被非特异性降解，这与前体到达线粒体中被阻断的加工酶后几乎立即被降解的观点一致。蛋白酶抑制剂对pCPS的合成几乎没有影响。这是通过分离多核糖体氨基苯甲脒来确定的；在存在多肽链起始抑制剂金精三羧酸的情况下，这两种多核糖体制剂在体外合成pCPS方面的活性大致相同。
