Purification and initial characterization of the proteasome from the higher plant Spinacia oleracea.
The proteasome (multicatalytic protease complex), a high molecular weight protein complex, has been purified from spinach leaves by successive chromatography on DEAE-cellulose, Bio-Gel A-1.5m, DEAE-TOYOPEARL 650C, and DEAE-5PW. The molecular mass was estimated to be 850 kDa by gel filtration. Polyacrylamide gel electrophoresis of the proteasome gave a single protein band under nondenaturing conditions and at least 10 bands in the range of 21-32 kDa in the presence of sodium dodecyl sulfate. By electron microscopy after negative staining with uranyl acetate, the proteasome from spinach appeared as symmetrical ring-shaped particles. The substrate specificity of proteasomes indicates that they contain at least three types of activity, namely, chymotrypsin-like, Staphylococcus aureus V8 protease-like, and trypsin-like activities. The former two activities were enhanced by poly-L-lysine or sodium dodecyl sulfate. Moreover, we examined the immunological reactivities of proteasomes from various eukaryotes. As a result, cross-immunoreactivities of some subunits were observed. These properties of the proteasome are similar to those of proteasomes isolated from various other eukaryotic sources.
Ozaki
M
M
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Japan.
Fujinami
K
K
Tanaka
K
K
Amemiya
Y
Y
Sato
T
T
Ogura
N
N
Nakagawa
H
H
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism / Q000648:ultrastructure; D010944:Plants / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D012967:Sodium Dodecyl Sulfate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex