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Catalytic and physico‐chemical characteristics of goat spleen cathepsin B
Author(s) -
Agarwal Sudhir K.,
Choudhury Santanu D.,
Lamsal Madhav,
Khan M. Yahiya
Publication year - 1997
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549700203681
Subject(s) - chemistry , chromatofocusing , cathepsin b , size exclusion chromatography , sephadex , molecular mass , biochemistry , enzyme , microbiology and biotechnology , cathepsin d , chromatography , cysteine , cathepsin , biology
To improve the level of purity of cathepsin B, we have modified the published procedure [Agarwal, S.K. and Khan, M.Y. (1987) Biochem. Int. 15, 785‐792] by incorporating CM‐Sephadex ion exchange chromatography and chromatofocusing. The enzyme thus isolated could be resolved into one 26 kDa major and a minor 27 kDa protein bands on SDS‐PAGE. The two components, however, could not be separated by gel filtration and they eluted, in a single peak corresponding to a molecular mass of 28.1 kDa . Among the various substrates tested, Z‐Phe‐Arg‐MCA with a Km of 0.058 mM and hemoglobin with a Km of 1.449 μM were the most preferred synthetic and protein substrates respectively. It was found to be a glycoprotein with an acidic pI of 4.8. The enzyme was activated by various thiol‐reducing reagents and inhibited by cysteine proteinase inhibitors, divalent cations, lysyl group modifiers, anti‐inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. Immunodiffusion studies with anti‐goat cathepsin B indicated a tissue/species dependence.