scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

scholarly journals Studies on cathepsin B in human articular cartilage

1978 ◽  
Vol 171 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M T Bayliss ◽  
S Y Ali
Keyword(s):  
Articular Cartilage ◽  
Cathepsin B ◽  
Cathepsin D ◽  
Human Articular Cartilage ◽  
Normal Cartilage ◽  
Elderly Individuals ◽  
Age Related ◽  
Hydrolysis Of ◽  
Increased Activity ◽  
Thiol Proteinase

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.

scholarly journals Preparation of cathepsins B and H by covalent chromatography and characterization of their catalytic sites by reaction with a thiol-specific two-protonic-state reactivity probe. Kinetic study of cathepsins B and H extending into alkaline media and a rapid spectroscopic titration of cathepsin H at pH 3-4

1985 ◽  
Vol 227 (2) ◽  
pp. 511-519 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Thiol Group ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Cysteine Proteinases ◽  
Acidic Media ◽  
Active Centre ◽  
Ph Values ◽  
Cathepsin H ◽  
Covalent Chromatography

A procedure for the isolation of cathepsin B (EC 3.4.22.1) and of cathepsin H from bovine spleen involving covalent chromatography by thiol-disulphide interchange and ion-exchange chromatography was devised. The stabilities of both cathepsins in alkaline media are markedly temperature-dependent, and reliable kinetic data can be obtained at pH values up to 8 by working at 25 degrees C with a continuous spectrophotometric assay. Both enzyme preparations contain only one type of thiol group as judged by reactivity characteristics towards 2,2′-dipyridyl disulphide at pH values up to 8; in each case this thiol group is essential for catalytic activity. Cathepsin H was characterized by kinetic analysis of the reactions of its thiol group with 2,2′-dipyridyl disulphide in the pH range approx. 2-8 and the analogous study on cathepsin B [Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] was extended to include reaction at pH values up to approx. 8. Cathepsin H, like the other cysteine proteinases, was shown to contain an interactive catalytic-site system in which the nucleophilic character of the sulphur atom is maintained in acidic media. The considerable differences in catalytic site characteristics detected by this two-protonic-state reactivity probe between cathepsin B, cathepsin H, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) are discussed. Reaction with 2,2′-dipyridyl disulphide in acidic media, which is known to provide a rapid spectrophotometric active centre titration for many cysteine proteinases, is applicable to cathepsin H. This is useful because other active-centre titrations have proved unsuitable in view of the relatively low reactivity of the thiol group in cathepsin H.

scholarly journals Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence

2004 ◽  
Vol 380 (3) ◽  
pp. 627-633 ◽  
Author(s):  
Daniel ROCHU ◽  
Nathalie VIGUIÉ ◽  
Frédérique RENAULT ◽  
David CROUZIER ◽  
Marie-Thérèse FROMENT ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Metal Cation ◽  
Active Site ◽  
Ph Dependence ◽  
Conformational Stability ◽  
Maximum Activity ◽  
Active Centre ◽  
Optimum Activity ◽  
Maximum Stability ◽  
Activation Phase

Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T≤35 °C, followed by an activation phase near 45–48 °C and then inactivation which was completed at 60 °C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different Tm values of 60.5–67 °C, 58–64 °C and 53–64 °C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

scholarly journals Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain

1984 ◽  
Vol 219 (3) ◽  
pp. 727-733 ◽  
Author(s):  
P Schack ◽  
N C Kaarsholm
Keyword(s):  
Ionic Strength ◽  
Thiol Group ◽  
Peptide Inhibitor ◽  
Affinity Column ◽  
Sh Group ◽  
The One ◽  
Hydrolysis Of ◽  
Nitrophenyl Ester ◽  
Thiol Proteinase ◽  
Immobilized Peptide

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.

scholarly journals Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa

1982 ◽  
Vol 205 (1) ◽  
pp. 147-152 ◽  
Author(s):  
S Shenolikar ◽  
K J Stevenson
Keyword(s):  
Gel Filtration ◽  
Extracellular Fluid ◽  
Thiol Group ◽  
Thermophilic Fungus ◽  
Sedimentation Equilibrium ◽  
Amino Acid Residues ◽  
Humicola Lanuginosa ◽  
Mercuric Ions ◽  
Thiol Proteinase

An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns. The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions. The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium. The H. lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues. This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates. This enzyme represents a unique example of a thiol proteinase obtained from a fungal source.

scholarly journals Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes

1976 ◽  
Vol 153 (2) ◽  
pp. 397-402 ◽  
Author(s):  
A Koj ◽  
J Chudzik ◽  
A Dubin
Keyword(s):  
Methyl Ester ◽  
Substrate Specificity ◽  
Rose Bengal ◽  
Ph Dependence ◽  
Maximum Activity ◽  
Enzyme Molecule ◽  
Active Centre ◽  
Pancreatic Elastase ◽  
Horse Blood ◽  
Nitrophenyl Ester

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.

scholarly journals Isolation, Purification, And Characterization Of Some Cystein Proteases From Bovine Mastites Milk

2009 ◽  
Vol 3 (1) ◽  
pp. 85-97
Author(s):  
K.S. Doosh
Keyword(s):  
Cathepsin B ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Mastitis ◽  
Deae Cellulose ◽  
Cysteine Proteases ◽  
Enzyme Characterization ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Molecular Weights

Proteolytic activity of cysteine proteases were studied in bovine mastitis milk, four fractions designated as F1,F2,F3,F4 with cysteine protease activity were separated from leukocytes cell by ion- exchange Chromatography through DEAE-Cellulose The most active fraction F4 was selected for further purification utilizing gel filtration Chromatography on Sephadex G-100 column it has been found that F4 most likely being cathepsin B. purification folds and the enzyme yield was 46.66 and 31.81% respectively . polyacrylamide gel electrophoresis test indicated that the enzyme has been purified to homogeneity by giving a single band . The results of enzyme characterization showed that the molecular weights were 31000 and 30000 Daltons as determined by gel filtration and electrophoresis methods in present of reducing agent SDS- PAGE respectively. The optimum pH for the enzyme activity was 6.0 and it was stable at pH values ranged between 4.5 - 6.5. The enzyme exhibited the maximum activity at 45ºC and the enzyme retained its entire activity over 30 min incubation at 30 -50 C and it retained (50, 20, 10) % of its entire activites over 30 min incubation at (60, 70, 80) C respectively. From this results and results observed from the effect of inhibitor and activator reagents we suggest that enzyme F4 possibly belonged to cathepsin B.

scholarly journals Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites

1986 ◽  
Vol 238 (1) ◽  
pp. 103-107 ◽  
Author(s):  
F Willenbrock ◽  
K Brocklehurst
Keyword(s):  
Cathepsin B ◽  
Cysteine Proteinase ◽  
Ph Dependence ◽  
Thiol Group ◽  
Ion Pair ◽  
Catalytic Site ◽  
Alkaline Media ◽  
Weakly Alkaline ◽  
Stem Bromelain ◽  
Ph Range

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.

The Specificity of Penicillopepsin

1971 ◽  
Vol 49 (10) ◽  
pp. 1134-1149 ◽  
Author(s):  
G. Mains ◽  
Miho Takahashi ◽  
J. Šodek ◽  
T. Hofmann
Keyword(s):  
Amino Acid ◽  
Glutamic Acid ◽  
Ph Dependence ◽  
Random Copolymer ◽  
The Other ◽  
High Rate ◽  
Hydrophobic Amino Acid ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Very High

The specificity of penicillopepsin was investigated with a number of different substrates. In agreement with earlier work (1), no action was observed on di- and tripeptide and ester substrates. However, the pepsin substrate carbobenzoxyglycylglycylphenylalanylphenylalanine 3-(4-pyridyl)propyl-1 ester was hydrolyzed between the two phenylalanine residues. No cleavage of homopolymers of glycine, alanine, and glutamic acid and the random copolymer of glutamic acid and tyrosine was observed. Polymers of lysine, lysine and tyrosine (2:1), and lysine, glutamic acid, and tyrosine (13:20:1) were, however, hydrolyzed giving mainly tripeptides to pentapeptides. Polylysine hydrolysis showed a pH dependence centering about a group of pK between 3 and 4. In glucagon and the S-sulfo B-chain of insulin, penicillopepsin hydrolyzed the same peptide bonds as the other acidic proteases, including pepsin and rennin, with very few exceptions. There are, however, distinct differences between the action of penicillopepsin and that of other proteases of low specificity. Peptide bonds which have a hydrophobic amino acid in the P1′ position (as defined by Berger and Schechter, Ref. 2) are preferentially cleaved by penicillopepsin. A kinetic study of the hydrolysis of bovine serum albumin showed very high rate constants for the initial cleavages. The present study shows the requirement for an extended binding site in penicillopepsin.

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