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Free keywords:
Adenosine Triphosphate/me [Metabolism]; Cell Fractionation; Centrifugation, Density Gradient; Cysteine Endopeptidases/ip [Isolation & Purification]; *Cysteine Endopeptidases/me [Metabolism]; Cysteine Endopeptidases/ul [Ultrastructure]; Image Enhancement; Isoenzymes; Lyases/me [Metabolism]; Multienzyme Complexes/ip [Isolation & Purification]; *Multienzyme Complexes/me [Metabolism]; Multienzyme Complexes/ul [Ultrastructure]; *Plant Leaves/en [Enzymology]; Proteins/me [Metabolism]; *Spinach/en [Enzymology]; Substrate Specificity; Support, Non-U.S. Gov't; Ubiquitins/me [Metabolism]
Abstract:
The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellulose, gel filtration on Biogel A-1.5, and glycerol density gradient centrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiquitin. By nondenaturing electrophoresis, the purified enzyme was separated into two distinct forms of the 26 S complex, named 26 S alpha and 26 S beta proteasomes, with different electrophoretic mobilities. The 26 S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115 kDa, which were thought to be those of a 20 S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and deubiquitinating activities, respectively. The subunit multiplicity of the spinach 26 S proteasome closely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multicomponent polypeptide complex. Electron microscopy showed that the 26 S proteasome complex had a "caterpillar"-like shape, consisting of four central protein layers, assumed to be the 20 S proteasome, with asymmetric V-shaped layers at each end. These structural and functional characteristics of the spinach 26 S proteasome showed marked similarity to those of the mammalian 26 S proteasomes reported recently, suggesting that the 26 S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.
