BN-PAGE在棕色脂肪线粒体呼吸链超级复合体分析中的应用

Detection of brown adipocyte mitochondrial respiratory chain supercomplexes by blue native polyacrylamide gel electrophoresis

目的建立一种敏感、特异的棕色脂肪线粒体超级复合体(supercomplexes,SCs)分离及活性检测方法,有助于了解棕色脂肪线粒体的功能。方法分离并纯化小鼠棕色脂肪线粒体。采用蓝色非变性聚丙烯酰胺凝胶电泳(blue native polya-crylamide gel electrophoresis,BN-PAGE)分离不同大小的线粒体SCs。经考马斯亮蓝染色和免疫印迹分析各组分的表达情况。将凝胶置于不同的底物液孵育,根据显色反应确定线粒体复合物的酶活性。结果成功提取棕色脂肪线粒体。考马斯亮蓝染色和免疫印迹结果显示棕色脂肪线粒体SCs清晰可见,分子量在720~1236 kD之间,与预期相符。棕色脂肪线粒体SCs在复合物Ⅰ或复合物Ⅱ的底物液中呈紫色,在复合物Ⅳ的底物液中呈棕色。结论本研究建立的棕色脂肪线粒体SCs分析方法简便易行、结果直观,为肥胖及其相关代谢性疾病的研究提供了实验基础。

Objective To establish a sensitive and specific method for isolating brown adipocyte mitochondrial supercomplexes(SCs)and detecting their activities,which will help to understand the function of brown adipocyte mitochondria.Methods Mitochondria were isolated and purified from brown adipose tissues in mice.SCs were separated using blue native polyacrylamide gel electrophoresis(BN-PAGE),and analyzed by Coomassie brilliant blue staining and immunoblotting.The gel was incubated in different substrate solutions,and then the enzymatic activity of the mitochondrial complex was determined based on the chromogenic reaction.Results Mitochondria was successfully isolated.The results of Coomassie brilliant blue staining and immunoblotting showed that the mitochondrial SCs with molecular weights of 720-1236 kD were clearly visible,which was consistent with expectations.The mitochondrial SCs presented purple in the substrate solution of complexⅠor complexⅡ,and brown in the substrate solution of complexⅣ.Conclusion The method for analyzing brown adipocyte mitochondrial SCs is simple,and the results are intuitive,which provides an experimental basis for the study of obesity and related metabolic diseases.