Jänsch L, Kruft V, Schmitz U K, Braun H P
Institut für Genbiologische Forschung GmbH, Berlin, Germany.
Eur J Biochem. 1995 Mar 15;228(3):878-85.
Analysis of cytochrome c reductase from potato by Tricine/SDS/PAGE reveals 10 bands representing 10 different subunits. In comparison to glycine/SDS/PAGE one additional small protein becomes visible, whereas the three large core proteins are not resolved. The identity of the subunits was determined by immunoblotting and direct sequence determination. Sequence data for the novel small component were used to derive oligonucleotides for probing a potato cDNA-library and isolating corresponding clones. The newly identified subunit is a 6.7-kDa protein, that exhibits significant sequence similarity to a 8.5-kDa subunit of cytochrome c reductase from yeast and the 6.5-kDa iron-sulfur-protein-binding factor from the equivalent enzyme complex from beef. Also the potato 6.7-kDa subunit can be dissociated from the cytochrome c reductase complex together with the iron-sulfur protein. To address the question of whether three or two core subunits occur simultaneously in monomeric cytochrome c reductase complexes from potato, a peptide-specific antibody was generated. The antiserum is capable of discriminating between the 55-kDa and 53-kDa core proteins, which can be separated by glycine/SDS/PAGE and which were previously found to be structurally related. Immunoprecipitations of isolated cytochrome c reductase from potato using this antibody revealed an enzyme complex containing only two core proteins. The simultaneous occurrence of only two core subunits was confirmed by a comparison of the molecular masses of cytochrome c reductase from potato and beef by blue-native-gel electrophoresis. Hence the cytochrome c reductase complexes from potato, beef and yeast have a very conserved subunit composition. The evolutionary implications of these findings are discussed.
通过Tricine/SDS/PAGE对马铃薯细胞色素c还原酶进行分析，发现有10条带，代表10种不同的亚基。与甘氨酸/SDS/PAGE相比，可看到一个额外的小蛋白，而三种大的核心蛋白未被分离。通过免疫印迹和直接测序确定了亚基的身份。利用新发现的小成分的序列数据推导寡核苷酸，用于探测马铃薯cDNA文库并分离相应的克隆。新鉴定的亚基是一种6.7 kDa的蛋白质，与酵母细胞色素c还原酶的8.5 kDa亚基以及牛肉中同等酶复合物的6.5 kDa铁硫蛋白结合因子具有显著的序列相似性。马铃薯的6.7 kDa亚基也可以与铁硫蛋白一起从细胞色素c还原酶复合物中解离出来。为了解决在马铃薯单体细胞色素c还原酶复合物中同时存在三个还是两个核心亚基的问题，制备了一种肽特异性抗体。该抗血清能够区分55 kDa和53 kDa的核心蛋白，这两种蛋白可以通过甘氨酸/SDS/PAGE分离，并且之前发现它们在结构上相关。用这种抗体对从马铃薯中分离的细胞色素c还原酶进行免疫沉淀，结果显示该酶复合物只含有两个核心蛋白。通过蓝色非变性凝胶电泳比较马铃薯和牛肉细胞色素c还原酶的分子量，证实了只同时存在两个核心亚基。因此，马铃薯、牛肉和酵母的细胞色素c还原酶复合物具有非常保守的亚基组成。讨论了这些发现的进化意义。
