Structural and functional characterization of 20S and 26S proteasomes from bovine brain.
Two proteins were isolated, in a stable form, from bovine brain by ion exchange chromatography, gel filtration and ultracentrifugation on glycerol gradient. They were identified as 20S and 26S proteasomes on the basis of molecular mass, migration velocity on non-denaturing gels, immunoreactivity, multipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins. However, the 26S proteasome has some properties not yet described for its counterpart of other tissues and from brain of this and other species. In particular, the ATP concentration required by the 26S proteasome to reach maximal peptidase activity was approximately 40-fold lower than the one required for maximal proteolytic activity on polyubiquitinylated substrates. Moreover, plots of substrate concentration vs. velocity gave a saturation curve for the 26S proteasome only, which, for the trypsin-like and post-glutamyl peptide hydrolase activities fitted the Michaelis-Menten equation, whereas for the chymotrypsin-like activity indicated multibinding site kinetics with positive cooperativity (n = 2.32+/-0.38). As concerns the 20S proteasome, its electrophoretic pattern on native gel revealed a single protein band, a feature, to our knowledge, not yet described for the brain particle of any species.
Piccinini
M
M
Dipartimento di Medicina e Oncologia Sperimentale, Sezione di Biochimica, Via Michelangelo n. 27/B, 10126, Torino, Italy.
Tazartes
O
O
Mostert
M
M
Musso
A
A
DeMarchi
M
M
Rinaudo
M T
MT
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007106:Immune Sera; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D046988:Proteasome Endopeptidase Complex; D014461:Ultracentrifugation
D007106:Immune Sera; D009097:Multienzyme Complexes; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease