scholarly journals Binding of proteinases to human α2-macroglobulin with its thioester bonds cleaved by methylamine in the presence of a thiol-group-cyanylating reagent

1985 ◽  
Vol 231 (2) ◽  
pp. 451-457 ◽  
Author(s):  
I Björk
Keyword(s):  
Conformational Change ◽  
Major Change ◽  
Thiol Group ◽  
Spectroscopic Properties ◽  
Structural Features ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Bean Trypsin Inhibitor ◽  
Similar Mode ◽  
The Stability

After cleavage of the thioester bonds of human alpha 2-macroglobulin (alpha 2M) by methylamine, the inhibitor undergoes an extensive conformational change and loses its ability to bind proteinases. In contrast, similar cleavage in the presence of dinitrophenyl thiocyanate, a reagent that cyanylates the liberated thiol groups, does not change the mobility of alpha 2M in gel electrophoresis, and the inhibitor also retains activity [Van Leuven, Marynen, Cassiman & Van den Berghe (1982) Biochem. J. 203, 405-411]. Analyses in this work show that also the spectroscopic properties of alpha 2M are essentially unperturbed under these conditions. These observations are consistent with the major change of the conformation of the protein having been arrested by the cyanylation reaction. However, several functional properties of the protein are altered, indicating that a limited conformational change does occur. The apparent stoichiometry of binding of trypsin is thus decreased to about 0.5 mol of enzyme/mol of alpha 2M. Nevertheless trypsin induces a similar conformational change in all molecules of the modified inhibitor as that induced in untreated alpha 2M. This behaviour indicates a similar mode of binding of the enzyme to the modified alpha 2M as to intact alpha 2M, but also a high extent of non-productive activation of binding sites in the modified inhibitor. A further difference to untreated alpha 2M is that most of the bound trypsin molecules react considerably faster with soya-bean trypsin inhibitor. The rate of inhibition of thrombin is also greatly decreased, and the modified inhibitor is more sensitive than untreated alpha 2M to proteolysis at sites outside the ‘bait’ region. The properties of the cyanylated human alpha 2M are thus similar to those of bovine alpha 2M in which the thioester bonds have been cleaved by methylamine in the absence of the cyanylating reagent [Björk, Lindblom & Lindahl (1985) Biochemistry 24, 2653-2660]. These results indicate that the thioester bonds of human and bovine alpha 2M are not required as such for the stability of the gross conformation of the protein or for the binding of proteinases. Nevertheless they participate directly in maintaining certain structural features, similar in the two inhibitors, that are necessary for full proteinase-binding ability. Disruption of these structures leads to a slower and less efficient trapping of the enzymes.

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 Interaction of uranium(VI) towards glutathione – an example to study different functional groups in one molecule

2011 ◽  
Vol 1 (1) ◽  
pp. 357-362
Author(s):  
L. Frost ◽  
Gerhard Geipel ◽  
K. Viehweger ◽  
G. Bernhard
Keyword(s):  
Stability Constant ◽  
Fluorescence Spectroscopy ◽  
Thiol Group ◽  
Spectroscopic Properties ◽  
Laser Fluorescence ◽  
Fluorescent Label ◽  
Binding Potential ◽  
Complex Stability ◽  
Vis Spectroscopy ◽  
The Stability

Abstract Glutathione, the most abundant thiol compound of the cell, has a great binding potential towards heavy metal ions. Hence it might influence the distribution of actinides on a cellular level. The unknown strength of the interaction of uranium(VI) with glutathione at physiologically relevant pH is subject of this paper and was studied with UV-vis spectroscopy and time-resolved laser-induced fluorescence spectroscopy (TRLFS). The complex stability constant of UO2H2GS+, logβ 0 121, was calculated to be 39.09 ± 0.15 and 39.04 ± 0.02 in case of UV-vis spectroscopy and TRLFS respectively. Therefore the average formation constant for UO2 2+ + H2GS− = UO2H2GS+ can be assigned to be log K 0 11 = 19.83 ± 0.15. Furthermore it was demonstrated that derivatization of the ligand associated with an enhancement of the ligand's spectroscopic properties can be used for the determination of complex stability constants and to assess the coordination chemistry in more detail. Using UV-vis spectroscopy, the stability constant of the complex between UO2 2+ and glutathione pyruvate S-conjugate, a well absorbing ligand in contrast to glutathione, was calculated to be >39.24 ± 0.08. Furthermore the interaction of UO2 2+ with glutathione derivatized with the fluorescent label monobromobimane was examined with femtosecond laser fluorescence spectroscopy. Thereby the stability constant of the 1:1 complex was determined to be >39.35 ± 0.02. Although the thiol group of glutathione was blocked a strong coordination was found. Thus a significant involvement of the thiol group in the coordination of U(VI) can be excluded.

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.

Functional characterization and structural modelling of peptidoglycan degrading β-N-acetyl-glucosaminidase from a dental isolate of Serratia marcescens

2020 ◽  
Vol 23 ◽  
Author(s):  
Aditi Rathee ◽  
Anil Panwar ◽  
Seema Kumari ◽  
Sanjay Chhibber ◽  
Ashok Kumar
Keyword(s):  
Functional Characterization ◽  
Major Change ◽  
Crystal Form ◽  
Bound State ◽  
Dynamics Simulation ◽  
Gram Positive ◽  
Structural Cell ◽  
Stability Structure ◽  
Arginine Residues ◽  
The Stability

Introduction:: Enzymatic degradation of peptidoglycan, a structural cell wall component of Gram-positive bacteria, has attracted considerable attention being a specific target for many known antibiotics. Methods:: Peptidoglycan hydrolases are involved in bacterial lysis through peptidoglycan degradation. β-N-acetylglucosaminidase, a peptidoglycan hydrolase, acts on O-glycosidic bonds formed by N-acetylglucosamine and N-acetyl muramic acid residues of peptidoglycan. Aim of present study was to study the action of β-N-acetylglucosaminidase, on methicillin- resistant Staphylococcus aureus (MRSA) and other Gram-negative bacteria. Results:: We investigated its dynamic behaviour using molecular dynamics simulation and observed that serine and alanine residues are involved in catalytic reaction in addition to aspartic acid, histidine, lysine and arginine residues. When simulated in its bound state, the RMSD values were found lesser than crystal form in the time stamp of 1000 picoseconds revealing its stability. Structure remained stably folded over 1000 picoseconds without undergoing any major change further confirming the stability of complex. Conclusion:: It can be concluded that enzymes belonging to this category can serve as a tool in eradicating Gram-positive pathogens and associated infections.

scholarly journals Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

2021 ◽  
pp. 100879
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Khang Diep ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
...  
Keyword(s):  
Protease Inhibitors ◽  
Bait Region ◽  
Α2 Macroglobulin

scholarly journals Redox-State Dependent Spectroscopic Properties of Porous Organic Polymers Containing Furan, Thiophene, and Selenophene

2017 ◽  
Vol 70 (11) ◽  
pp. 1227 ◽  
Author(s):  
Carol Hua ◽  
Stone Woo ◽  
Aditya Rawal ◽  
Floriana Tuna ◽  
James M. Hook ◽  
...  
Keyword(s):  
Solid State ◽  
Redox State ◽  
Spectroscopic Properties ◽  
Structural Features ◽  
Organic Polymers ◽  
Stimuli Responsive ◽  
Porous Organic Polymers ◽  
Paramagnetic Resonance ◽  
Responsive Materials ◽  
State Dependent

A series of electroactive triarylamine porous organic polymers (POPs) with furan, thiophene, and selenophene (POP-O, POP-S, and POP-Se) linkers have been synthesised and their electronic and spectroscopic properties investigated as a function of redox state. Solid state NMR provided insight into the structural features of the POPs, while in situ solid state Vis-NIR and electron paramagnetic resonance spectroelectrochemistry showed that the distinct redox states in POP-S could be reversibly accessed. The development of redox-active porous organic polymers with heterocyclic linkers affords their potential application as stimuli responsive materials in gas storage, catalysis, and as electrochromic materials.

FABRICATION OF NANOPOROUS CHITOSAN MEMBRANES

NANO ◽  
2010 ◽  
Vol 05 (01) ◽  
pp. 53-60 ◽  
Author(s):  
XIAOLIANG WANG ◽  
XIANG LI ◽  
ELEANOR STRIDE ◽  
MOHAN EDIRISINGHE
Keyword(s):  
Low Frequency ◽  
Drug Carriers ◽  
Structural Features ◽  
Wound Dressings ◽  
Ftir Analysis ◽  
Tissue Engineering Scaffolds ◽  
Nanoscale Structures ◽  
Low Frequency Ultrasound ◽  
Chitosan Membranes ◽  
The Stability

Naturally derived biopolymers have been widely used for biomedical applications such as drug carriers, wound dressings, and tissue engineering scaffolds. Chitosan is a typical polysaccharide of great interest due to its biocompatibility and film-formability. Chitosan membranes with controllable porous structures also have significant potential in membrane chromatography. Thus, the processing of membranes with porous nanoscale structures is of great importance, but it is also challenging and this has limited the application of these membranes to date. In this study, with the aid of a carefully selected surfactant, polyethyleneglycol stearate-40, chitosan membranes with a well controlled nanoscale structure were successfully prepared. Additional control over the membrane structure was obtained by exposing the suspension to high intensity, low frequency ultrasound. It was found that the concentration of chitosan/surfactant ratio and the ultrasound exposure conditions affect the structural features of the membranes. The stability of nanopores in the membrane was improved by intensive ultrasonication. Furthermore, the stability of the blended suspensions and the intermolecular interactions between chitosan and the surfactant were investigated using scanning electron microscope and Fourier transform infrared spectroscopy (FTIR) analysis, respectively. Hydrogen bonds and possible reaction sites for molecular interactions in the two polymers were also confirmed by FTIR analysis.

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