We purified the 20S proteasome from the alga Chara corallina Willd with DEAE-ion-exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme by nondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two-dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin-like, trypsin-like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin-like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin-like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin-like activity was inhibited by N-ethylmaleimide, p-chlaromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin-like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.
