PURIFICATION AND PROPERTIES OF RABBIT MUSCLE PROTEASOME, AND ITS EFFECT ON MYOFIBRILLAR STRUCTURE

This paper describes the purification and properties of a multicatalyt ic proteinase complex, proteasome, from rabbit skeletal muscle, and it s effect on myofibrillar structure. The purified proteasome gave a sin gle band on polyacrylamide gel electrophoresis under nondenaturing con ditions and gave eight bands under denaturing conditions, indicating t hat this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data i ndicate that the purified proteasome is not 26S, but 20S. The proteaso me degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0 , activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5. 0 still retained about 30 similar to 60% of the activity at pH 8.0, in dicating the possibility that the proteasome is active in the muscle d uring conditioning. When the proteasome was heated at 60 degrees C for 20 min and treated in the presence of 0.005% SDS, the activity increa sed over 1.5 and 4.5 times, respectively. SDS remarkably increased the V-max value of the enzyme at pH 8.0. The proteasome was also activate d by high hydrostatic pressure up to 100 similar to 150 MPa and gradua lly decreased at 200 MPa or higher. Electron microscopic observation r evealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by pro teasome. (C) 1998 Elsevier Science Ltd. All rights reserved.