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.

scholarly journals Relation of internal thioesters to conformational change and receptor-recognition site in α2-macroglobulin complexes

1982 ◽  
Vol 203 (2) ◽  
pp. 405-411 ◽  
Author(s):  
F Van Leuven ◽  
P Marynen ◽  
J J Cassiman ◽  
H Van den Berghe
Keyword(s):  
Monoclonal Antibody ◽  
Conformational Change ◽  
Trypsin Inhibitor ◽  
Soya Bean ◽  
Recognition Site ◽  
Thiol Groups ◽  
Α2 Macroglobulin ◽  
Receptor Recognition ◽  
Complex Receptor ◽  
Bean Trypsin Inhibitor

The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M. The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated. Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M. Other thiol-specific reagents were less effective. When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active. Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes. The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.

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 Binding of anhydrotrypsin to α2-macroglobulin

1980 ◽  
Vol 189 (2) ◽  
pp. 255-261 ◽  
Author(s):  
C A Sayers ◽  
A J Barrett
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Soya Bean ◽  
Α2 Macroglobulin ◽  
Bean Trypsin Inhibitor

Radiolabelled anhydrotrypsin was bound by alpha 2M (alpha 2-macroglobulin) sufficiently tightly to resist separation during gel electrophoresis; 2 mol of anhydrotrypsin were bound/mol of alpha 2M, but the interaction differed in important respects from that between active proteinases and alpha 2M. Anhydrotrypsin was bound by the electrophoretically ‘fast’ form of alpha 2M, although much less effectively than by the ‘slow’ form. The inactive enzyme was displaced from alpha 2M by trypsin inhibitor, the order of effectiveness being aprotinin > soya-bean trypsin inhibitor > benzamidine. Saturation of alpha 2M with anhydrotrypsin did not prevent subsequent binding and inhibition of active trypsin by the alpha 2M, and the anhydrotrypsin was not displaced during this reaction. Anhydrotrypsin bound by alpha 2M retained its ability to react with antibodies against trypsin, whereas bound trypsin did not.

scholarly journals Ligand binding, conformational change and plasma elimination of human, mouse and rat α-macroglobulin proteinase inhibitors

1983 ◽  
Vol 209 (1) ◽  
pp. 99-105 ◽  
Author(s):  
S L Gonias ◽  
A E Balber ◽  
W J Hubbard ◽  
S V Pizzo
Keyword(s):  
Ligand Binding ◽  
Conformational Change ◽  
Trypsin Inhibitor ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Soya Bean ◽  
The Other ◽  
Complex Reaction ◽  
Bean Trypsin Inhibitor ◽  
Esterolytic Activity

Rat alpha 1-macroglobulin (alpha 1M), rat alpha 2-macroglobulin (alpha 2M) migrated as single bands on non-denaturing gels when purified by the methods described. All three proteins demonstrated increased mobility after reaction with trypsin. A single saturable pathway rapidly cleared complexes of trypsin and the alpha-macroglobulins of mouse, rat and human from the circulation of mice. None of the native alpha-macroglobulins competed for clearance with the trypsin complexes. [14C]Methylamine incorporation was 4.1, 3.9, 2.6 and 3.2 mol/mol of proteinase inhibitor for human alpha 2M, rat alpha 1M, rat alpha 2M and mouse alpha 2M, respectively. Only rat alpha 2M, the acute-phase alpha-macroglobulin studied, showed no evidence of conformational change when subjected to electrophoresis after reaction with methylamine. The clearance of rat alpha 2M-methylamine was comparable with that of the native molecule. The other alpha-macroglobulin-methylamine complexes cleared faster than the inhibitors that had not reacted. Rat alpha 2M and rat alpha 2M-methylamine bound equivalent quantities of 1251-labelled trypsin (1.01 and 0.96 mol/mol respectively). The soya-bean trypsin inhibitor-resistant esterolytic activity of trypsin bound to rat alpha 2M-methylamine was approx. 90% suppressed compared with proteinase bound to native rat alpha 2M. This suppression was not due to a change in the affinity of soya-bean trypsin inhibitor for the complex. Reaction of rat alpha 2M-methylamine with trypsin resulted in a ‘slow’ to ‘fast’ electrophoretic conversion of the proteinase inhibitor, and exposure of the signal on the alpha 2M that causes the complex to clear from the murine circulation.

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 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 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.

Measurement of Endotoxin Levels in Plasma Using Limulus Amoebocyte Lysate and Chromogenic Substrate, S2222

1979 ◽  
Author(s):  
S Clark ◽  
M Scully ◽  
P Webb ◽  
V Kakkar
Keyword(s):  
Trypsin Inhibitor ◽  
Final Concentration ◽  
Soya Bean ◽  
Lag Phase ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Linear Calibration ◽  
Limulus Amoebocyte Lysate ◽  
Bean Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor

A method has been devised for the measurement of endotoxin in plasma using the chromogenic substrate, S2222, a substrate which has been shown to be particularly sensitive to the Limulus Lysate. Time curves of the rate of release of the chromogen in mixtures in which procoagulase activation was concurrent, were complex with a lag phase which was shortened by increasing endotoxin concentrations. At a final concentration of 0.5ng/ml and 370 activation was complete within 60 minutes. The enzyme was inhibited by soya bean trypsin inhibitor but not by pancreatic trypsin inhibitor or hirudin. In the method finally adopted the lysate (25µl) was incubated with endotoxin (E.coli 026.86 Difco) and magnesium chloride (final concentration 33mM) in a total volume of 225µl. After 12 minutes preincubation 165µl of S2222(0.4mM) was added and the increase in abdorbance at 405nm over two minutes measured using an Abbott Biochromatic Analyser 100. Linear assay curves were obtained with final concentration of 0.2 to 2.0ngs endotoxin/ml with ΔOD 405/min of 0.35 at 2.0ng endotoxin/ml of final incubation mixture. ΔOD /min in control tubes were of the order of 0.02. For measurement from plasma samples, endotoxin was first extracted with chloroform. Linear calibration curves were achieved at a concentration of endotoxin of 1 to 5ng/ml of whole blood with a net OD/min at the highest concentration of 0.25.

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