Purification and characterization of the mitochondrial translocase from Euglena gracilis.
Purification and characterization of the mitochondrial translocase from Euglena gracilis.
复制标题
纤细眼虫线粒体转位酶的纯化和表征。
DOI：
10.1016/0003-9861(85)90793-3
复制
发表时间：
1985
影响因子：
3.9
通讯作者：
Spremulli,LL
中科院分区：
文献类型：
--
作者：
Eberly,SL;Spremulli,LL
利用梭链孢酸亲和层析法和传统的层析技术，经四步纯化得到纯度大于50%的纤细眼虫线粒体蛋白生物合成延伸因子G（EF-Gmt）。该纯化方案导致1100倍纯化，从全细胞提取物制备的核糖体后上清液中存在的总EF-G活性的回收率约为3%。gracilisEF-Gm的分子量约为76，000，与原核易位酶的分子量相当。与已研究过的其他易位酶一样，E. gracilisEF-Gmi N-乙基马来酰亚胺导致聚合活性的损失，表明必需的半胱氨酸残基在催化活性中的作用。GDP部分保护EF-Gmt免受N-乙基马来酰亚胺的失活。gracilisEF-Gmt对大肠杆菌和E.对叶绿体核糖体有活性，但对小麦胚胞质核糖体的活性很低。在这方面，它与酵母的线粒体移位酶显着不同，酵母的线粒体移位酶对叶绿体核糖体的活性很小。当测定一个E。coliribosomes、E. gracilisEF-Gm对类固醇抗生素夫西地酸敏感，其水平与灭活E. coliEF-G.它比E敏感。gracil是叶绿体EF-G，比Bacillus subtilus EF-G敏感。当测定一个E。尽管失活所需的夫西地酸的确切水平略有改变，但观察到了同样的敏感性趋势。
TheEuglena gracilismitochondrial protein biosynthetic elongation factor G (EF-Gmt) has been purified in four steps to greater than 50% homogeneity by use of a fusidic acid affinity procedure and conventional Chromatographic techniques. The purification scheme results in 1100-fold purification with about 3% recovery of the total EF-G activity present in the postribosomal supernatant prepared from whole cell extracts.E. gracilisEF-Gmthas an approximate molecular weight of 76,000, comparable to that observed for procaryotic translocases. As is the case for other translocases which have been examined, pretreatment ofE. gracilisEF-GmtwithN-ethylmaleimide results in a loss of polymerization activity, indicating a role for an essential cysteine residue in catalytic activity. GDP partially protects EF-GmtfromN-ethylmaleimide inactivation.E. gracilisEF-Gmtfunctions well on bothEscherichia coliandE. gracilischloroplast ribosomes, but has negligible activity on wheat germ cytoplasmic ribosomes. In this respect, it differs significantly from the mitochondrial translocase of yeast which has very little activity on chloroplast ribosomes. When assayed onE. coliribosomes,E. gracilisEF-Gmtis sensitive to the steroid antibiotic, fusidic acid, at levels similar to that required for inactivation ofE. coliEF-G. It is less sensitive thanE. gracilischloroplast EF-G, and is more sensitive thanBacillus subtilusEF-G. When assayed onE. gracilischloroplast ribosomes, the same trends in sensitivities are observed, although the exact level of fusidic acid required for inactivation is slightly altered.