Mills N C, Ray D B, Littlejohn R A, Horst I A, Kowal J
Anal Biochem. 1984 Apr;138(1):164-80. doi: 10.1016/0003-2697(84)90785-1.
The requirements for in vitro mitochondrial protein synthesis have been studied using isolated mitochondria from cultured adrenal Y-1 tumor cells from mice. By reducing the reaction volume to 50 microliter we were able to assay in replicate the requirements for various reaction components using trichloroacetic acid (TCA)-precipitable counts for a quantitative evaluation with time of incubation. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography was also used for a qualitative and quantitative evaluation of the translation products. With the optimized system, 1 to 3% of added [35S]methionine was incorporated. The products of mitochondrial protein synthesis range from 70,000 to 5000 molecular weight. Major autoradiographic bands were observed at 38,000, 31,000, 23,000, 20,000, and 5600 molecular weight as separated on 10 to 20% gradient SDS-polyacrylamide gels; however, 20 to 30 protein products of various molecular weights were discernible. Mitochondrial concentrations of 0.8 to 1.4 mg/ml of incubation gave the better incorporation of [35S]methionine per milligram of protein. Total [35S]methionine incorporated into mitochondrial protein was greatest at 25 degrees C after 90 min. Chloramphenicol at 10 micrograms/ml inhibited mitochondrial protein synthesis by more than 50% and at 100 micrograms/ml inhibited incorporation by more than 95%. Cycloheximide had no effect on incorporation at less than 1.0 mg/ml. Magnesium and ATP in a molar ratio of one to one at 5 mM gave optimal incorporation. Other energy generating systems using oxidative phosphorylation to supply ATP for protein synthesis were not as effective as ATP and 5 mM phosphoenol pyruvate, 20 micrograms/ml pyruvate kinase and 5 mM a-ketoglutarate. In contrast to in vitro yeast mitochondrial protein synthesis, no enhancement of in vitro adrenal cell mitochondrial protein synthesis was found with GTP or its analogs. The buffers N,N-bis(2-hydroxyethyl)glycine, N-(tris(hydroxymethyl)methyl)glycine, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid were superior to Tris-HCl for mitochondrial protein synthesis. Optimal pH for [35S]methionine incorporation into mitochondrial proteins was pH 7.0 to 7.6. Potassium at 50 to 90 mM gave the best incorporation of [35S]methionine, and the higher molecular weight products of translation were enhanced at these concentrations. Sodium at 10 to 40 mM had no effect; however, 100 mM sodium inhibited label incorporation by 30%. Calcium at 100 microM inhibited mitochondrial protein synthesis by approximately 50%, and at 1.0 mM little if any incorporation occurred.(ABSTRACT TRUNCATED AT 400 WORDS)
利用从小鼠培养的肾上腺Y-1肿瘤细胞中分离出的线粒体，对体外线粒体蛋白质合成的需求进行了研究。通过将反应体积减少到50微升，我们能够使用三氯乙酸（TCA）沉淀计数法对各种反应成分的需求进行重复测定，以便随着孵育时间进行定量评估。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳后进行放射自显影，也用于对翻译产物进行定性和定量评估。在优化的系统中，添加的[35S]甲硫氨酸有1%至3%被掺入。线粒体蛋白质合成的产物分子量范围为70,000至5000。在10%至20%梯度SDS-聚丙烯酰胺凝胶上分离时，在38,000、31,000、23,000、20,000和5600分子量处观察到主要的放射自显影条带；然而，可以分辨出20至30种不同分子量的蛋白质产物。孵育液中线粒体浓度为0.8至1.4毫克/毫升时，每毫克蛋白质对[35S]甲硫氨酸的掺入效果更好。90分钟后，在25摄氏度时，掺入线粒体蛋白质中的总[35S]甲硫氨酸量最大。10微克/毫升的氯霉素抑制线粒体蛋白质合成超过50%，100微克/毫升时抑制掺入超过95%。低于1.0毫克/毫升时，环己酰亚胺对掺入没有影响。镁和ATP以1:1的摩尔比、5毫摩尔浓度时，掺入效果最佳。其他利用氧化磷酸化提供ATP用于蛋白质合成的能量产生系统，不如ATP以及5毫摩尔磷酸烯醇丙酮酸、20微克/毫升丙酮酸激酶和5毫摩尔α-酮戊二酸有效。与体外酵母线粒体蛋白质合成不同，未发现GTP或其类似物能增强体外肾上腺细胞线粒体蛋白质合成。缓冲液N,N-双（2-羟乙基）甘氨酸、N-（三（羟甲基）甲基）甘氨酸和N-2-羟乙基哌嗪-N'-2-乙磺酸在用于线粒体蛋白质合成时优于Tris-HCl。[35S]甲硫氨酸掺入线粒体蛋白质的最佳pH值为7.0至7.6。50至90毫摩尔的钾能使[35S]甲硫氨酸的掺入效果最佳，在这些浓度下，较高分子量的翻译产物会增加。10至40毫摩尔的钠没有影响；然而，100毫摩尔的钠会使标记掺入减少30%。100微摩尔的钙抑制线粒体蛋白质合成约50%，1.0毫摩尔时几乎没有掺入发生。（摘要截短至400字）
