PURIFICATION AND CHARACTERIZATION OF THE 26-S PROTEASOME FROM SPINACHLEAVES

The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiq uitin-ligated proteins was purified from spinach leaves 60 near homoge neity 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, lysozym es in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiq uitin. By nondenaturing electrophoresis, the purified enzyme was separ ated 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 mo lecular 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 a ssociated 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 m ulticomponent 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, wit h asymmetric V-shaped layers at each end. These structural and functio nal characteristics of the spinach 26 S proteasome showed marked simil arity to those of the mammalian 26 S proteasomes reported recently, su ggesting 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.