大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法

Protein synthesis and characterization in isolated mitochondria from rat brain

Aim: To establish a system of mitochondrial translation in vitro of rat brain and identify it's production of protein by molecular weight.Methods:Mitochondria isolated from hemishere of rat brain by differential centrifugations.The optimization of mitochondrial translation in vitro by 3H-Lencine incorporation was explored. 35S-methionine labled products of mitochondrial protein synthesis were identified by SDS-PAGE and fluorogaphy.Results:Isolated mitochondria had a highly activity oxidative phosphorylation and respiratory control ratio(RCR) was between 3.5 and 5.5.The activity of 3H-Lencine incorporation in isolated mitochondria in vitro increased with time of incubation in 60 min and maintained a steady level.The maximal activity of 3H-Lencine incorporation per milligram mitochondria protein occured at 1 mg mitochondria/ml of incubation mix.The major auto radiographic bands could be observed at 86,68,56,43,33,29,25 and 18(kD) molecular weight separated on SDS-PAGE.Conclusion:The translation system of rat brain mitochondria in vitro is faithful and high activity,can be used to study mtDNA expression and regulation in mammalian brain at the level of translation.