20S Proteasome Complex from Thermoplasma volcanium

Kocabıyık, Semra
In eucaryotes, 26S proteasome complex is the central enzyme of ubiquitin dependent protein degradation in the cytosol and nucleus. 26S complex is composed of two components: 20S catalytic core and 19S complex exhibiting ATPase activity. The simplest form of proteasomes is 20S proteasome which has been found in many archaeal species and bacteria (especially in G + actinomycetes). This arcaebacterial enzyme is composed of only 2 types of subunits, designated α and β that are so organized that α-subunits form 2 outer rings and β-subunits form two inner rings of a barrel shaped structure.In this project we attempted at detection and isolation of proteasome from a thermophilic archaea Thermoplasma volcanium and carried out its biochemical and genetic characterizations. The proteasome was initially detected on 0.5% gelatin containing SDS-polyacrylamide gel as a 150-kDa proteolytic activity which was confirmed by immunoblotting using T. acidophilum 20S proteasome specific antibody. Purification of proteasome related activities involved a two-step ammonium sulfate precipitation. The 40-80% fraction was loaded on a SephacrylS-300 gel filtration chromatography column, and fractions showing 'chymotryptic' activity with Ala-Ala-Phe-pNA, were loaded on ion-exchange column prepared with DEAE Sepharose Fast Flow. Proteins were eluted with an NaCl gardient (0-500 mm). Then, proteasome containing fractions were directly loaded onto a hydroxylapatide affinity column. With the isolated proteasome preparation besides chymotryptic activity, peptidylglutamyl peptide hydrolase activity also, was detected with CBZ-Leu-Leu-Glu-β-NA, at pH 7.5 and 60 °C. The enzyme activity was enhanced several fold by Mg2+ and Ca2+. The serine proteinase inhibitors irreversibly inhibited the activity.
Citation Formats
S. Kocabıyık, “20S Proteasome Complex from Thermoplasma volcanium,” presented at the 27th Meeting of the Federation of European Biochemical Societies (30 Haziran - 05 Temmuz 2001), Lisbon, Portugal, 2001, Accessed: 00, 2021. [Online]. Available: https://hdl.handle.net/11511/72518.