Isolation and characterisation of cathepsin-B from bovine pancreas (original) (raw)
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Isolation and characterization of cathepsin D from human gastric mucosa
Collection of Czechoslovak Chemical Communications, 1981
The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.
On the tissue/species dependence of cathepsin B isozymes
Molecular and cellular biochemistry, 1997
Characterization of cathepsin B from buffalo kidney and goat spleen showed the presence of isozymes in case of the goat spleen (GSCB-I and GSCB-II) whereas cathepsin B from buffalo kidney exhibited only one form (BKCB). The molecular weights determined by SDS-PAGE for GSCB-I, GSCB-II, and BKCB were 25.7, 26.6 and 25.5 kDa respectively. The kinetic parameters (Km and Vmax) of GSCB-I showed close similarities with BKCB against alpha-N-benzoyl-DL-arginine-2-napthylamide whereas GSCB-II was closer to the buffalo enzyme with regards to its activity against Z-Arg-Arg-MCA and Z-Phe-Arg-MCA. All the three enzymes had similar sensitivities towards urea, antipain and leupeptin. However, clear differences were observed in the inhibition patterns of the enzyme with iodoacetic acid and iodoacetamide. Differences in the kinetic, immunogenic and some catalytic properties of GSCB-I and II, which had similarities with regard to most of their physico-chemical properties, were considered to be due to ...
Purification and amino acid sequence of chicken liver cathepsin L
Biochemistry, 1987
Biochemistry 1987, 26, 5689-5695 5689 the extent that a comparison of the lysed and intact cell systems is valid, there are several possible explanations for this discrepancy. It is possible that the level of 5-HPETE generated is sufficient to deplete glutathione, the principal reducing agent for peroxides. This is unlikely, however, because the intracellular concentration of glutathione is about 5 mM (Egan & Gale, 1985) and the total intracellular concentration of 5lipoxygenase products from endogenous arachidonic acid reaches only about 0.08 mM (Sun & McGuire, 1984; calculated assuming 5 X lo8 cells/g wet weight). It is possible that the 5-HPETE that is formed in intact cells is segregated from the peroxidases so that the rapid rate of reduction of 5-HPETE in vitro is not indicative of the in vivo rate. It is also possible that the intrinsic partitioning of 5-HPETE on the enzyme between dissociation and conversion to LTA, is different for 5-lipoxygenase in its native intracellular state and the relative inefficiency observed in vitro is an artifact. We cannot at present provide evidence for either of these possibilities.
Catalytic and physico-chemical characteristics of goat spleen cathepsin B
IUBMB Life, 1997
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 K of 0.058 mM and hemoglobin with a K of 1.449 ~tM were the most m 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, a_nti-inflammatory drug and denaturing agents. The hydrodynamic behaviour of cathepsin B suggested a compact and globular conformation. !mmunodiffusion studies with anti-goat cathepsin B indicated a tissue/ species dependence.
Purification and some properties of buffalo spleen cathepsin B
Journal of Biosciences, 1989
Purification of cathepsin Β from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffaloenzyme is similar to cathepsin Β from other tissues with respect to these properties.
Purification and characterization of cathepsin B from goat brain
Journal of Biosciences
Cathepsin L-like proteinase was purified ~1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH 4 ) 2 SO 4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be ~65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mβNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-β-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mβNA was hydrolyzed maximally by the enzyme (K m value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mβNA, Z-Phe-Val-Arg-4mβNA, Z-Arg-Arg-4mβNA and Z-Ala-Arg-Arg-4mβNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and β-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with K i value 44 × 10 -9 M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg 2+ , Ca 2+ , Cu 2+ , Li 2+ , K + , Cd 2+ , Ni 2+ , Ba 2+ , Mn 2+ , Co 2+ and Sn 2+ also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40ºC. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mβNA was ~50-55ºC with an activation energy E a of ~6.34 KCal mole -1 .
Studies on activation and inhibition of cathepsin B from buffalo liver
Journal of Protein Chemistry, 1996
Cathepsin B (EC3.4.22.1) was purified from buffalo liver. The enzyme activity against a-benzoyl-DL-arginine-naphthylamine (BANA) was substantially reduced by heat (above 37~ and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-l-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu 2+ (20-200/zM) and Ca 2+ (30-250mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), and p-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.
Primary structure of bovine cathepsin S Comparison to cathepsins L, H, B and papain
FEBS Letters, 1991
The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (J-CR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3' untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and I3 are 56%, 47% and 31% respectively.
Biological Chemistry, 1999
We established a continuous semi-microassay, and for large-scale studies both a stopped and a continuous microtiter plate assay for the fluorometric determination of cathepsin L and cathepsin S activities in body fluids, tissues or cell extracts in the presence of cathepsin B. For the detection of enzymatic activities we used the synthetic substrate Z-Phe-Arg-AMC, and for discrimination between cathepsin L, S and cathepsin B the specific inhibitor CA-074 for blocking interfering cathepsin B activities was applied. Furthermore, we took advantage of the stability of cathepsin S at pH 7.5 for further differentiation between cathepsin L and cathepsin S activities. The kinetic assays were characterized in terms of imprecision, analytical sensitivity, accuracy and substrate concentration. The within-run coefficients of variation were found to be 4.9%–7.2% for the continuous semi-microassay, 10.3%–11.7% for the stopped, and 4.5%–11.8% for the continuous microtiter plate assay. The between-...
Problems associated with the assay of cathepsin D in meat and meat products
Food Chemistry, 1991
Cathepsin D is usually assayed by following the release of the trichloroacetic (TCA)-soluble peptides from denatured haemoglobin at 280nm, but some artefacts may appear giving false results. Cathepsin D activity has therefore been assayed under different conditions in muscle, liver and dry-cured ham extracts. Substantial errors (around 50-56 %) become evident when using the classical standard assay. The assay of cathepsin D activity in muscle extracts should include the use of a blank containing a specific inhibitor such as isovaler ylpepstatin.