20S Proteasome Complex from Thermoplasma volcanium

Date

2001-06-30

Author

Kocabıyık, Semra

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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.

URI

https://hdl.handle.net/11511/72518

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Department of Biology, Conference / Seminar