title: Pitfalls in invertebrate proteasome assays.
abstract: The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.
authors.affiliation: Alfred Wegener Institute for Polar and Marine Research, Functional Ecology, 27570 Bremerhaven, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Götze
authors.forename: Aneesh
authors.initials: A
authors.lastname: Bose
authors.forename: Doris
authors.initials: D
authors.lastname: Abele
authors.forename: Inna M
authors.initials: IM
authors.lastname: Sokolova
authors.forename: Reinhard
authors.initials: R
authors.lastname: Saborowski
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D002918:Chymotrypsin / Q000378:metabolism; D003445:Crustacea / Q000201:enzymology*; D057075:Enzyme Assays; D006434:Hemocytes / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009842:Oligopeptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000378:metabolism; D059748:Proteolysis
chemical_list: D009842:Oligopeptides; D061988:Proteasome Inhibitors; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin