Sodium dodecyl sulfate (sds) activation of the 20s proteasome in rat liver

Toru Shibatani, Walter F. Ward

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

Abstract

The peptidase activity of the 20S proteasome (multi-catalytic 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.

Original languageEnglish (US)
Pages (from-to)160-166
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume321
Issue number1
DOIs
StatePublished - Aug 1 1995

Keywords

  • Activation
  • Chymotrypsin-like
  • Fluorogenic substrate
  • Immunoprecipitation
  • Multicatalytic protease complex
  • Peptidase
  • Peptidylglutamyl peptide-hydrolyzing activity
  • Proteasome
  • SDS
  • Trypsin-like

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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