Sodium dodecyl sulfate (SDS) activation of the 20S proteasome in rat liver.
The peptidase activity of the 20S proteasome (multicatalytic protease complex) was examined in the 100,000g supernatant fraction prepared from rat liver tissue. Fluorogenic substrates for three proteasome peptidase activities were selected on the basis of (i) observation of an accelerated degradation in the presence of sodium dodecyl sulfate (SDS) and (ii) preferential degradation by the proteasome. Peptidase activities were assayed using an immunoprecipitation technique utilizing polyclonal antibodies raised against the purified rat proteasome. The ability to demonstrate SDS activation of the proteasome is shown to be dependent upon the choice of substrate. In addition, among the cytosolic peptidases, the property of SDS activation appears to be unique to the proteasome. SDS activation profiles were determined for each peptidase activity. Chymotrypsin-like and peptidylglutamyl peptide-hydrolyzing activities exhibit a broad plateau of activation between 0.04 and 0.05% SDS. Trypsin-like activity exhibits a sharp peak of activation at an SDS concentration of 0.04%. The SDS activation profile can be altered by changing the protein (proteasome) concentration, i.e., increasing protein (proteasome) concentration of the reaction mixture produces a marked rightward shift of the activation profile. On the other hand, changing the substrate concentration does not alter the profile. In conclusion, a technique for measuring proteasome peptidase activity in the 100,000g supernatant has been described. This approach increases the ease of measurement of peptidase activity and provides data which may more closely reflect the in vivo activity of the proteasome.
Shibatani
T
T
Department of Physiology, University of Texas Health Science Center at San Antonio 78284-7756, USA.
Ward
W F
WF
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D007106:Immune Sera; D007074:Immunoglobulin G; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011916:Rats, Inbred F344; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D007106:Immune Sera; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009842:Oligopeptides; D012967:Sodium Dodecyl Sulfate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex