PURIFICATION AND CHARACTERIZATION OF ACTIVE PROTEASOME (26S PROTEASOME) FROM GOLDFISH OVARIES

The cytosol fraction (150,000 xg supernatant) of goldfish ovarian homogenate hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA in the absence of SDS. The use of a stepwise gradient and ATP prevented the loss of this activity during purification. Active proteasome was purified to homogeneity from ovarian cytosol using five steps of chromatography. The purified active proteasome had chymotrypsin-, trypsin-, and V8 protease-like activities even in the absence of SDS. The enzyme exhibited two bands on native PAGE. Electrophoresis and Western blot analyses showed that the enzyme consisted of at least 15 protein components ranging in molecular mass from 35.5 to 140 kDa, as well as the multiple subunits of the latent proteasome ranging in molecular mass from 23.5 to 31.5 kDa. The molecular weight and sedimentation coefficient of the active proteasome were estimated to be 1,200 kDa and 29.4S, respectively, both of which are larger than those of the latent proteasome of the same species. In electron micrographs, the active proteasome appeared as a dumbbell-like image. It is concluded that the active proteasome purified from goldfish oocyte cytosol is identical to the 26S proteolytic complex reported in various eukaryotic cells.