Catalytic and physico-chemical characteristics of goat spleen cathepsin B.
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 microM 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.
Agarwal
S K
SK
Department of Biochemistry, Dr. Ram Manohar Lohia Avadh University, Faizabad, India.
Choudhury
S D
SD
Lamsal
M
M
Khan
M Y
MY
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002401:Cathepsin B / Q000737:chemistry* / Q000494:pharmacology / Q000502:physiology*; D002852:Chromatography, Ion Exchange / Q000379:methods; D003429:Cross Reactions; D004789:Enzyme Activation; D005634:Fructose-Bisphosphate Aldolase / Q000187:drug effects / Q000378:metabolism; D006041:Goats; D007700:Kinetics; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000737:chemistry / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology
D000596:Amino Acids; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; D002401:Cathepsin B; D005634:Fructose-Bisphosphate Aldolase