Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates.
The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson's disease, Alzheimer's disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific.
Rodgers
Kenneth J
KJ
Cell Biology Unit, The Heart Research Institute, 145 Missenden Road, Camperdown, NSW 2050, Sydney, Australia. k.rodgers@hri.org.au <k.rodgers@hri.org.au>
Dean
Roger T
RT
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D009842:Oligopeptides; D025801:Ubiquitin; C067713:lactacystin; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine; C078846:epoxomicin