Purification and characterization of cytoplasmic and proteasome associated chymotrypsin-like proteases from Thermoplasma volcanium

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Tezin Türü: Doktora

Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Edebiyat Fakültesi, Biyolojik Bilimler Bölümü, Türkiye

Tezin Onay Tarihi: 2003

Öğrenci: İNCİ ÖZDEMİR

Danışman: SEMRA KOCABIYIK

Özet:

In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 æmoles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (a-subunit) and 21.9 kDa (β-subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The