Characterization of active proteasome (26S proteasome) involved in Bufo japonicus oocyte maturation

It is known that the cytosol fraction (105,000 Xg supernatant) of Bufo japo nicus oocytes hydrolyzes Suc-Leu-Leu-Val-Tyr-MCA in the absence of an activ ator (SDS). In the present study, the active proteasome was purified from t he cytosol fraction by sequential column chromatographies. The molecular ma ss of the purified active proteasome was estimated to be 990 kDa. Electroph oresis and Western blot analyses showed that the active proteasome has at l east 25 protein components ranging in molecular mass from 23.5 to 125 kDa. The purified proteasome possessed chymotrypsin-like, trypsin-like and pepti dylglutamyl peptidase activities even in the absence of SDS. The chymotryps in-like activity was inhibited by diisopropyl fluorophosphate (DFP) at the same concentration that prevents oocyte maturation of Bufo. Phosphorylation analysis showed that 56 (p56) and 125 (p125) kDa proteins were phosphoryla ted by a protein kinase co-purified in the active proteasome fraction. It i s concluded that the active proteasome involved in the regulation of oocyte maturation in Bufo is the 26 S proteasome and its function may be regulate d by phosphorylation of p125.