Mori M, Miura S, Tatibana M, Cohen P P
Proc Natl Acad Sci U S A. 1979 Oct;76(10):5071-5. doi: 10.1073/pnas.76.10.5071.
Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.
大鼠肝脏的总RNA或聚腺苷酸（+）RNA在兔网织红细胞或小麦胚芽蛋白合成系统中进行翻译，然后通过间接免疫沉淀法分离合成的氨甲酰磷酸合成酶I [氨甲酰磷酸合成酶（氨）；二氧化碳：氨连接酶（ADP形成，氨基甲酰磷酸化），EC 6.3.4.16]，该方法使用在酶结合的琼脂糖凝胶和金黄色葡萄球菌细胞上纯化的抗体。体外产物在十二烷基硫酸钠/聚丙烯酰胺凝胶上作为一种多肽移动，其分子量比成熟酶的亚基（160,000道尔顿）大约大5000道尔顿。通过直接免疫沉淀或双抗体沉淀法也获得了相同的多肽。成熟酶与体外产物有效地竞争与抗氨甲酰磷酸合成酶I抗体的相互作用。金黄色葡萄球菌蛋白酶对体外产物的消化产生了与成熟酶相似的肽片段模式。大鼠肝脏的线粒体膜制剂将体外产物转化为一种在十二烷基硫酸钠凝胶电泳上与成熟亚基迁移率相同的多肽。在大鼠肝脏的胞质溶胶或微粒体部分均未检测到类似的蛋白水解活性。这些结果表明该酶是以较大的前体形式合成的，通过翻译后加工转化为成熟形式的酶。
