Structural and functional characterization of 20S and 26S proteasomes frombovine brain

Two proteins were isolated, in a stable form, from bovine brain by ion exch ange chromatography, gel filtration and ultracentrifugation on glycerol gra dient. They were identified as 20S and 26S proteasomes on the basis of mole cular mass, migration velocity on non-denaturing gels, immunoreactivity, mu ltipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins, However, t he 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 pepti dase activity was approximately 40-fold lower than the one required for max imal proteolytic activity on polyubiquitinylated substrates. Moreover, plot s of substrate concentration vs. velocity gave a saturation curve for the 2 6S proteasome only, which, for the trypsin-like and post-glutamyl peptide h ydrolase activities fitted the Michaelis-Menten equation, whereas for the c hymotrypsin-like activity indicated multibinding site kinetics with positiv e cooperativity (n = 2.32 +/- 0.38), As concerns the 20S proteasome, its el ectrophoretic pattern on native gel revealed a single protein band, a featu re, to our knowledge, not yet described for the brain particle of any speci es. (C) 2000 Elsevier Science B.V. All rights reserved.