scholarly journals Procollagenase activator produced by rabbit uterine cervical fibroblasts

1987 ◽  
Vol 241 (2) ◽  
pp. 527-534 ◽  
Author(s):  
M Ishibashi ◽  
A Ito ◽  
K Sakyo ◽  
Y Mori
Keyword(s):  
Culture Medium ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Serine Proteinase ◽  
Active Species ◽  
Rabbit Plasma ◽  
Neutral Proteinase ◽  
Type Iv

Culture medium from rabbit uterine cervical fibroblasts contained a procollagenase and a neutral proproteinase which acts as a procollagenase activator. These two proenzymes have been purified by a combination of ion-exchange, affinity and gel chromatographies. The purified neutral proproteinase showed Mr 60,000 with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This neutral proproteinase was activated by trypsin, 4-aminophenylmercuric acetate (APMA) and plasmin, and the active species of the proteinase had Mr 53,000 when activated by APMA; kallikrein and urokinase did not activate this proproteinase. The purified neutral proteinase was inhibited by EDTA, 1,10-phenanthroline and rabbit plasma, but not by serine proteinase inhibitors, suggesting that this proteinase is a metal-dependent proteinase. The purified enzyme could also degrade gelatin, casein, proteoglycan and type IV procollagen. The purified procollagenase had Mr 55,000 and was activated by trypsin, APMA and the active neutral proteinase. These activations were accompanied by decrease in Mr, and the activated species had an Mr which was approx. 10,000 less than that of the procollagenase. In particular, procollagenase activation with neutral proteinase depended on incubation time and proteolytic activity of proteinase. These results indicate that activation of procollagenase by the rabbit uterine neutral proteinase is related to limited proteolysis in the procollagenase molecule.

scholarly journals Mouse macrophage elastase. Purification and characterization as a metalloproteinase

1981 ◽  
Vol 193 (2) ◽  
pp. 589-605 ◽  
Author(s):  
M J Banda ◽  
Z Werb
Keyword(s):  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
Serine Proteinases ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Predominant Form ◽  
Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

Purification and partial characterization of kininogenase activity from Schistosoma mansoni adult worms

Parasitology ◽  
1998 ◽  
Vol 117 (4) ◽  
pp. 311-319 ◽  
Author(s):  
W. S. CARVALHO ◽  
C. T. LOPES ◽  
L. JULIANO ◽  
P. M. Z. COELHO ◽  
J. R. CUNHA-MELO ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Adult Worm ◽  
Ethylenediaminetetraacetic Acid ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Rat Plasma ◽  
Male Adult ◽  
Arterial Blood

An enzyme presenting kallikrein-like activity (designated sK1) was purified from the supernatant of Schistosoma mansoni adult worm homogenate. The enzyme cleaves bradykinin from purified rat plasma kininogen. Activity was optimal at pH 9·0 and the enzyme showed amidolytic activity, since it hydrolysed the kallikrein synthetic substrate d-Pro-Phe-Arg-p-nitroanilide. The activity of sK1 upon rat plasma kininogen was strongly inhibited by the serine proteinase inhibitors phenylmethanesulfonyl fluoride, aprotinin or soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid or sodium tetrathionate. The molecular mass of sK1, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 66 kDa and the pI value, estimated by analytical chromatofocusing, was 4·2. Physical and chemical properties suggest that sK1 is a serine proteinase of the kallikrein family. Evidence is presented which suggests that sK1 is a component of the tegumental surface of the parasite and the levels of its activity in the male adult worm are approximately 21 times higher than those in the female adult worm. The intravenous injection of 3 μg of sK1 into an anaesthetized rat induced a drastic reduction in the arterial blood pressure of the animal. This effect lasted for about 1 min, and was followed by a progressive recovery of the arterial pressure. Neither bradycardia nor cardiac arrhythmias were noticed, suggesting a peripheral vasodilation effect. The presence of sK1 on the surface of adult male worms could play an important role in the wandering capacity of coupled worms into the visceral vasculature of the host.

scholarly journals Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

1979 ◽  
Vol 183 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Jean-Louis Azanza ◽  
Jacques Raymond ◽  
Jean-Michel Robin ◽  
Patrick Cottin ◽  
André Ducastaing
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Rabbit Skeletal Muscle ◽  
Neutral Proteinase

Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α1-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl2, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150–2158]. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase.

scholarly journals The inactivation of native enzymes by a neutral proteinase from rat intestinal muscle

1978 ◽  
Vol 173 (1) ◽  
pp. 291-298 ◽  
Author(s):  
R J Beynon ◽  
J Kay
Keyword(s):  
Smooth Muscle ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Serine Proteinase ◽  
Partial Purification ◽  
Intestinal Smooth Muscle ◽  
Bean Trypsin Inhibitor ◽  
Ph Range

1. The solubilization and partial purification of a proteinase from the intestinal smooth muscle of rats fed on protein-free diets are described. 2. It has a mol.wt. of about 33000 and it is stable over a narrow pH range. 3. From its susceptibility to known modifers of proteolytic enzymes, it appears to be a serine proteinase of a trypsin-like nature. Active-site titration with soya-bean trypsin inhibitor shows that the concentration of proteinase was about 3 microgram/g wet wt. of intestinal smooth muscle. However, the muscle proteinase demonstrates a marked ability for inactivating enzymes in their native conformation at neutral pH. It is about 100 times more efficient than pancreatic trypsin when the inactivating activities are compared on an approximately equimolar basis. 4. Inactivation of the substrate enzymes is accompanied by limited proteolysis, as demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 5. An endogenous inhibitor was separated from the proteinase by fractionation with (NH4)2SO4. 6. Contamination of the muscle tissue by lumen, mucosal or blood proteinases and inhibitors is shown to be unlikely. 7. A role for the neutral trypsin-like proteinase in initiating the degradation of intracellular enzymes is considered.

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

Transport and proteolytic processing of rabbit cardiac cathepsin D

1985 ◽  
Vol 248 (1) ◽  
pp. C135-C144
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
S. W. Worobec ◽  
M. Lesch
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteolytic Processing ◽  
Time Dependent ◽  
Low Density ◽  
Enzyme Maturation

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

Catabolism of streptokinase and polyethylene glycol-streptokinase: evidence for transport of intact forms through the biliary system in the mouse

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 73-79 ◽  
Author(s):  
FH Brucato ◽  
SV Pizzo
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Gel Electrophoresis ◽  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gi Tract ◽  
Substantial Fraction ◽  
Sds Page ◽  
Derivatives Of

Abstract The catabolism of streptokinase (SK) and polyethylene glycol derivatives of SK (PEG-SK) were studied in mice. The clearance and catabolism of SK:plasmin (SK:Pm) and PEG-SK:Pm activator complexes were also investigated. Native 125I-SK cleared rapidly (t1/2 = 15 minutes) from the circulation, with the majority of the ligand accumulating in the liver and gastrointestinal (GI) tract and a substantial fraction also localizing in the kidneys. SK, which was removed from the plasma by the liver, was secreted into bile and then the GI tract. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that 125I-SK recovered from liver and bile was homogeneous and of the same molecular weight (mol wt approximately 50,200) as native SK. PEG-125I-SK cleared slowly (t1/2 greater than 200 minutes), with more than 80% of the preparation localizing in liver and GI tract. The PEG-125I-SK secreted into the bile was also intact. The bile containing 125I-SK was incubated with stoichiometric amounts of plasminogen and electrophoresed under nondenaturing conditions. This study demonstrated that the secreted SK was able to form SK:Pg complexes. SDS-PAGE also showed activation of 125I-Pg that was incubated with recovered bile containing the SK. 125I-SK:Pm catabolism was also studied. In these experiments, the mol wt approximately 42,000 fragment obtained when SK is cleaved by plasmin was found in the bile. This fragment of 125I-SK was not recovered as part of a complex with plasmin, consistent with our previous observations that catabolism of SK:Pm involves transfer of the plasmin to plasma proteinase inhibitors while SK is catabolized independently. By contrast, when PEG-125I-SK:Pm was injected into mice, only intact PEG-125I-SK was found in the bile, consistent with our previous observations that the PEG derivatization blocks its degradation by plasmin.

scholarly journals Studies on the Production of Fungal Peroxidases inAspergillus niger

2000 ◽  
Vol 66 (7) ◽  
pp. 3016-3023 ◽  
Author(s):  
Ana Conesa ◽  
Cees A. M. J. J. van den Hondel ◽  
Peter J. Punt
Keyword(s):  
Lignin Peroxidase ◽  
Manganese Peroxidase ◽  
Culture Medium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limiting Factors ◽  
Efficient Production ◽  
Mrna Levels ◽  
Extracellular Medium

ABSTRACT To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

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