Structure and function of proteasome in higher plants

通过deae -纤维素层析、Biogel A-1.5m凝胶过滤和甘油密度梯度离心，从菠菜叶中纯化出催化atp依赖性泛素连接蛋白分解的26S蛋白酶体复合物，使其接近均匀性。纯化后的酶被证明能够以atp依赖的方式降解多泛素化溶菌酶，但并非未经修饰，同时atp酶活性为蛋白质水解提供能量，异肽酶活性产生游离泛素。通过非变性电泳，纯化后的酶被分离成不同形式的26S复合物，分别命名为26Salpha和26Sbeta蛋白酶体，具有不同的电泳迁移率。26S蛋白酶体由多个分子量为23-35和39-115kDa的多肽组成，这些多肽被认为是具有多催化蛋白酶活性的20S蛋白酶体的多肽，它们分别具有atp酶和去泛素化活性的相关调控部分。菠菜26S蛋白酶体的亚基多样性与大鼠肝脏的亚基多样性基本相似，在某些成分上差异较小。该多组分多肽复合物的组装过程中不含巯基键。电镜显示，26S蛋白酶体复合体呈“毛毛虫”状，由四个中心蛋白层组成，假设为20S蛋白酶体，两端各有不对称的v形层。菠菜26S蛋白酶体的这些结构和功能特征与最近报道的哺乳动物26S蛋白酶体具有明显的相似性，表明26S蛋白酶体广泛分布于真核细胞中，在催化可溶性能量和泛素依赖的蛋白水解途径中具有普遍的重要性。

The 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellurose, gel filtration on Biogel A-1.5m and glycerol density gradient cenrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozyme in an ATP-dependent fashion, coupled with ATPase activity supplying energy for proteolysis, and isopeptidase activity to generate free ubiquitin. By non-denaturing electrophoresis, the puried enzyme was separated into to distinct forms of the 26S complex, named 26Salpha and 26Sbeta proteasomes, with different electorophoretic mobilities. The 26S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115kDa, which were thought to be those of a 20S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and de-ubiquitinating activities, respectively. the subunit multiplicity of the spinach 26S proteasome dosely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multi-component polypeptide complex. Electron microscopy showed that the 26S proteasome complex had a "caterpillar" like shape, consisting of four central protein layrs, assumed to be the 20S proteasome, with asymmetric V-shaped layrs of each end. These structural and functional characteristics of the spinach 26S proteasome showed marked similarity to those of the mammalian 26S proteasomes reported recently, suggesting that the 26S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy-and ubiquitin-dependent proteolytic pathway.