Purification and biochemical characterization of native ERp29 from rat liver.
Purification and biochemical characterization of native ERp29 from rat liver.
复制标题
大鼠肝脏天然 ERp29 的纯化和生化特征。
作者：
M. Hubbard;Jonathan E. Mangum;N. Mchugh
ERp29是最近表征的ER（内质网）腔的居民，其具有广泛的生物学意义，在动物细胞中广泛且大量地表达。ERp29被认为是分泌性蛋白质的一种通用折叠助手，并可能作为一种PDI（蛋白质二硫化物异构酶）样分子伴侣发挥作用。在本论文中，我们报告了第一次纯化的同质性和直接功能分析的天然ERp29，这导致了意想不到的发现，ERp29缺乏PDI样折叠活动。ERp29在非变性条件下纯化4800倍，利用对肝素的不寻常亲和力。另外两个生化标志，将有助于ERp29同系物的分类，即特异质行为的ERp29的大小排阻色谱（M（r）单体质量）。与PDI和平行纯化的共同居民（钙网蛋白，ERp60）相比，天然ERp29缺乏经典的伴侣，二硫还原酶和异构酶，和钙结合活性。在伴侣蛋白测定中，ERp29既不保护底物蛋白质免于热聚集，也不与化学变性蛋白质稳定地相互作用，如通过交联检测到的。ERp29也没有表现出对钙网蛋白（伴侣）或PDI和ERp60（二硫还原酶）的辅助活性。通过反驳长期以来的预测伴侣活性，这些结果暴露ERp29作为一个功能独特的成员的ER机制，并提示修订的假设，ERp29作为一个非经典的折叠助手。本研究建立的天然制备和生化标志为解决ERp29功能性孤儿状态的持续努力提供了有用的基础。
ERp29 is a recently characterized resident of the ER (endoplasmic reticulum) lumen that has broad biological significance, being expressed ubiquitously and abundantly in animal cells. As an apparent housekeeper, ERp29 is thought to be a general folding assistant for secretory proteins and to probably function as a PDI (protein disulphide isomerase)-like molecular chaperone. In the present paper, we report the first purification to homogeneity and direct functional analysis of native ERp29, which has led to the unexpected finding that ERp29 lacks PDI-like folding activities. ERp29 was purified 4800-fold in non-denaturing conditions exploiting an unusual affinity for heparin. Two additional biochemical hallmarks that will assist the classification of ERp29 homologues were identified, namely the idiosyncratic behaviours of ERp29 on size-exclusion chromatography (M(r)monomeric mass). In contrast with PDI and parallel-purified co-residents (calreticulin, ERp60), native ERp29 lacked classical chaperone, disulphide reductase and isomerase, and calcium-binding activities. In the chaperone assays, ERp29 neither protected substrate proteins against thermal aggregation nor interacted stably with chemically denatured proteins as detected by cross-linking. ERp29 also did not exhibit helper activity toward calreticulin (chaperone) or PDI and ERp60 (disulphide reductase). By refuting long-standing predictions about chaperone activity, these results expose ERp29 as a functionally distinct member of the ER machinery and prompt a revised hypothesis that ERp29 acts as a non-classical folding assistant. The native preparation and biochemical hallmarks established here provide a useful foundation for ongoing efforts to resolve the functional orphan status of ERp29.
脱氢抗坏血酸还原酶的分光光度测定。
影响因子：
2.9
作者：
Stahl,RL;Liebes,LF;Farber,CM;Silber,R
通讯作者：
Silber,R
从人红细胞中纯化和表征谷胱甘肽依赖性脱氢抗坏血酸还原酶。
DOI：
10.1006/bbrc.1996.0555
发表时间：
1996
期刊：
Biochemical and biophysical research communications.
影响因子：
--
作者：
Xu,DP;Washburn,MP;Sun,GP;Wells,WW
通讯作者：
Wells,WW