scholarly journals Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes

1978 ◽  
Vol 173 (3) ◽  
pp. 949-958 ◽  
Author(s):  
Z Werb ◽  
J T Dingle ◽  
J J Reynolds ◽  
A J Barrett
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Filtration ◽  
Synovial Fibroblasts ◽  
Enzymic Activity ◽  
Polymorphonuclear Leucocyte ◽  
Exchange Chromatography ◽  
Soya Bean ◽  
Polymorphonuclear Leucocytes ◽  
Neutral Proteinase ◽  
Bean Trypsin Inhibitor

A neutral proteinase secreted by rabbit synovial fibroblasts in parallel with specific collagenase was partially purified by ion-exchange chromatography. At pH 7.6 this proteinase degraded 35S-labelled bovine nasal proteoglycan and azo-casein. The enzymic activity was inhibited by EDTA, 1,10-phenanthroline and serum, whereas di-isopropyl phosphorofluoridate and soya-bean trypsin inhibitor had little effect. By gel filtration the apparent mol.wt. of the enzyme was 25000. The fibroblast neutral proteinase was compared with the proteoglycan-degrading neutral proteinases of rabbit polymorphonuclear-leucocyte granules. Two distinct activities were found in the granules: one was inhibited by soya-bean trypsin inhibitor and the other by EDTA. The proteoglycan-degrading proteinases of rabbit fibroblasts and polymorphonuclear leucocytes at acid pH also were examined. Both cathepsin D and a thiol-dependent proteinase contributed to the degradation of proteoglycan at pH 4.5.

scholarly journals Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin

1985 ◽  
Vol 226 (2) ◽  
pp. 477-485 ◽  
Author(s):  
M Z Atassi
Keyword(s):  
Binding Site ◽  
Trypsin Inhibitor ◽  
Binding Activity ◽  
Enzymic Activity ◽  
Soya Bean ◽  
Free Form ◽  
Protein Substrates ◽  
Substrate Analogues ◽  
Bean Trypsin Inhibitor ◽  
Alpha 1 Antitrypsin

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.

scholarly journals Kinetics of native and modified Bowman–Birk soya-bean trypsin inhibitor on growth and enzymes activities of the chick pancreas

1979 ◽  
Vol 42 (1) ◽  
pp. 121-126 ◽  
Author(s):  
Z. Madar
Keyword(s):  
Trypsin Inhibitor ◽  
Enzymic Activity ◽  
Soya Bean ◽  
Enzymes Activities ◽  
Bean Trypsin Inhibitor ◽  
Inhibitory Site ◽  
Kinetics Of

1. The Bowman-Birk soya-bean trypsin inhibitor (BBTI) begins to cause pancreatic enlargement and increased enzymic activity in the pancreas of chicks after a minimum of 7 d of feeding.2. The active inhibitory site of BBTI against trypsin is the factor involved in the pancreatic enlargement and increase of pancreatic nzesyme activity in chicks.

scholarly journals Evidence for polymorphonuclear-leucocyte-derived proteinases in arthritic cartilage

1981 ◽  
Vol 193 (1) ◽  
pp. 193-202 ◽  
Author(s):  
J D Sandy ◽  
A Sriratana ◽  
H L Brown ◽  
D A Lowther
Keyword(s):  
Guanidine Hydrochloride ◽  
Apparent Molecular Weight ◽  
Joint Inflammation ◽  
Polymorphonuclear Leucocyte ◽  
Soya Bean ◽  
Polymorphonuclear Leucocytes ◽  
Cartilage Proteoglycan ◽  
Marked Inhibition ◽  
Bean Trypsin Inhibitor ◽  
Proteoglycan Degradation

1. An enzyme that degrades proteoglycan at neutral pH was extracted with 4 M-guanidine hydrochloride from the articular cartilage of rabbits with antigen-induced arthritis. 2. The enzyme had an apparent molecular weight on Ultrogel AcA 54 of about 8000 and was optimally active at pH 7.5 in Tris/HCl buffer containing 0.2 M-NaCl. The partially purified preparation was totally inhibited by 0.01 mM-N-acetyldialanylprolylvalylchloromethane, severely inhibited by 2 mM-phenylmethanesulphonyl fluoride and soya-bean trypsin inhibitor (200 microgram/ml) and slightly inhibited by 10 mM-EDTA. Marked inhibition was also obtained with a cytosolic fraction prepared from rabbit polymorphonuclear leucocytes. 3. All properties of the enzyme were virtually identical with those of an ‘elastase-like’ proteinase that was isolated from rabbit polymorphonuclear-leucocyte granules. 4. The results are consistent with the idea that cartilage proteoglycan degradation in acute joint inflammation is due at least partly to the diffusion into the cartilage of proteinases derived from synovial-fluid polymorphonuclear leucocytes.

Activation of latent collagenase from polymorphonuclear leukocytes by cathepsin G

1979 ◽  
Vol 44 (10) ◽  
pp. 3177-3182 ◽  
Author(s):  
Mária Stančíková ◽  
Karel Trnavský
Keyword(s):  
Affinity Chromatography ◽  
Trypsin Inhibitor ◽  
Polymorphonuclear Leukocytes ◽  
Soya Bean ◽  
Cathepsin G ◽  
Sepharose Column ◽  
Bean Trypsin Inhibitor ◽  
Human Polymorphonuclear Leukocytes

Cathepsin G was isolated from human polymorphonuclear leukocytes and purified by affinity chromatography on Antilysin-Sepharose column. Purified enzyme activated later collagenase isolated from leukocytes. Activation at 36°C was maximal after 30 min incubation. Inhibitors of cathepsin G - soya-bean trypsin inhibitor, diisopropyl phosphofluoridate and Antilysin were active in inhibiting the activation of latent collagenase by cathepsin G.

scholarly journals Purification and properties of a kininogenin from the venom of Vipera ammodytes ammodytes

1976 ◽  
Vol 153 (2) ◽  
pp. 409-414 ◽  
Author(s):  
G S Bailey ◽  
R A Shipolini
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Vasoactive Peptides ◽  
Bovine Trypsin ◽  
Purification And Properties ◽  
Vipera Ammodytes

A kininogenin (EC 3.4.21.8) was purified from the venom of Vipera ammodytes ammodytes (European sand viper) by a combination of gel filtration and ion-exchange chromatography. The enzyme is approximately six times more active than bovine trypsin in its ability to release vasoactive peptides from a plasma precursor. The kininogenin is a glycoprotein containing 18-20% by weight of carbohydrate. It showed a mol. wt. of 40500 on gel filtration. Gel electrophoresis of the reduced sample in the presence of sodium dodecyl sulphate and 2-mercaptoethanol revealed the presence of two major components of mol.wt. 34300 and 31300. The heterogeneity, which was also observed on disc electrophoresis, was removed by incubation with neuraminidase. After incubation with neuraminidase the kininogenin retained full enzymic activity and possessed an isoelectric point of pH7.2. The carbohydrate content has been decreased to 10% by weight, and the single component seen on electrophoresis in the presence of sodium dodecyl sulphate and 2-mercaptoethanol corresponded to a mol.wt. of 29500.

scholarly journals Partial purification and characterization of a novel neutral proteinase from human uterine cervix

1980 ◽  
Vol 185 (2) ◽  
pp. 443-450 ◽  
Author(s):  
Akira Ito ◽  
Hiroko Ihara ◽  
Yo Mori
Keyword(s):  
Uterine Cervix ◽  
Gel Filtration ◽  
Reaction System ◽  
Partial Purification ◽  
High Concentration ◽  
Neutral Proteinase ◽  
Bean Trypsin Inhibitor ◽  
Cervical Stroma ◽  
High Concentrations ◽  
Hydrolysed Casein

1. Human uterine cervical stroma was found to contain a Ca2+-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×105 by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.

scholarly journals SUBSTRATES OF HAGEMAN FACTOR

1974 ◽  
Vol 140 (6) ◽  
pp. 1615-1630 ◽  
Author(s):  
Louis W. Heck ◽  
Allen P. Kaplan
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lima Bean ◽  
Hageman Factor ◽  
Α2 Macroglobulin ◽  
Coagulant Activity ◽  
Bean Trypsin Inhibitor

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α1-chymotrypsin inhibitor, inter α-trypsin inhibitor, α2-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α1-antitrypsin, and it was absent in α1-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α1-antitrypsin.

An Endogenous Protease Activating Plasma Inactive Renin

1978 ◽  
Vol 55 (s4) ◽  
pp. 133s-134s ◽  
Author(s):  
B. J. Leckie
Keyword(s):  
Human Plasma ◽  
Serine Protease ◽  
Protease Inhibitors ◽  
Trypsin Inhibitor ◽  
Soya Bean ◽  
Acid Activation ◽  
Bean Trypsin Inhibitor ◽  
Inactive Renin ◽  
Inhibited Acid ◽  
Endogenous Protease

1. The protease inhibitors Trasylol and soya-bean trypsin inhibitor prevented the activation of plasma inactive renin by acid. 2. N-Ethylmaleimide inhibited acid-activation to some extent but o-phenanthroline had no effect. 3. Acid-activation of the inactive renin in human plasma is mediated by a serine protease.

scholarly journals Functional modifications of α2-macroglobulin by primary amines. Kinetics of inactivation of α2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin

1982 ◽  
Vol 201 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Fred Van Leuven ◽  
Jean-Jacques Cassiman ◽  
Herman Van Den Berghe
Keyword(s):  
Conformational Change ◽  
Trypsin Inhibitor ◽  
Binding Capacity ◽  
Soya Bean ◽  
Primary Amines ◽  
Slow Phase ◽  
Subunit Structure ◽  
Α2 Macroglobulin ◽  
Bean Trypsin Inhibitor ◽  
Kinetics Of

The unique steric inhibition of endopeptidases by human α2M (α2-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by α2M was closely correlated with the loss of the methylamine-reactive sites in α2M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/α2M ratios, no unaccounted loss of trypsin-binding capacity occurred. As α2M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form α2M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [14C]methylamine with α2M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of α2M with time of methylamine treatment. It was found that conformational change of α2M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of 125I-labelled trypsin to α2M did follow the same kinetics as the conformational change. This discrepancy between total binding (125I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound 125I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of α2M and to the mechanism of formation of the complex.

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