THE MULTICATALYTIC PROTEINASE (PROTEASOME) OF THE HAWKMOTH, MANDUCA-SEXTA - CATALYTIC PROPERTIES AND IMMUNOLOGICAL COMPARISON WITH THE LOBSTER ENZYME COMPLEX

The proteasome plays a central role in ubiquitin-dependent and -indepe ndent proteolysis in eukaryotic cells, The hawkmoth proteasome was pur ified from larval body wall and characterized with respect to substrat e specificity, sensitivity to protease inhibitors, and cross-reactivit y with monoclonal antibodies (mAbs) raised against human placenta prot easome. Leupeptin selectively inhibited the trypsin-like activity (T-L ) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activi ties, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglut amyl peptide hydrolase, branched-chain amino acid preferring, and case inolytic activities 20-, 18-, and 3.8-fold, respectively, All four pep tidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimen sional immunoblot analysis showed that the level and subunit compositi on of the proteasome varied between tissues, The relative levels of pr oteasome were high in intersegmental muscle and ovary, lower in Malpig hian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body, The tissues differed in the relative am ount of a 41-kDa doublet; a 22-kDa subunit was present only in the mal e accessory gland, Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, co mpared with 28 subunits in lobster, Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human ze ta, C2, C3, and C8 Lu-type subunits in the hawkmoth and lobster enzyme s, Two of the four mAbs reacted with three or more of the hawkmoth sub units and three of the mAbs reacted with two or more of the lobster su bunits, In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) o r 16 (hawkmoth) subunits were alpha-type, These results suggest that m uch of the subunit complexity of the arthropod proteasomes is a conseq uence of extensive post-translational modifications. (C) 1995 Academic Press, Inc.