scholarly journals Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives

1996 ◽  
Vol 318 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Hanne GRØN ◽  
Ida B. THØGERSEN ◽  
Jan J. ENGHILD ◽  
Salvatore V PIZZO
Keyword(s):  
Room Temperature ◽  
Elevated Temperatures ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Complement Components ◽  
Thiol Ester ◽  
Chemically Modified ◽  
Inhibitory Capacity ◽  
Α2 Macroglobulin ◽  
The Complement System

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.

scholarly journals α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester

2020 ◽  
Vol 295 (49) ◽  
pp. 16732-16742 ◽  
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
Ida B. Thøgersen ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Primary Amines ◽  
Hydroxyl Groups ◽  
Histidine Residue ◽  
Protease Inhibition ◽  
Complement Components ◽  
Proteolytic Activation ◽  
Thiol Ester ◽  
Thiol Esters ◽  
Α2 Macroglobulin

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

scholarly journals Limulus α2-macroglobulin. First evidence in an invertebrate for a protein containing an internal thiol ester bond

1987 ◽  
Vol 248 (3) ◽  
pp. 703-707 ◽  
Author(s):  
P B Armstrong ◽  
J P Quigley
Keyword(s):  
Proteinase Inhibitor ◽  
Thermal Denaturation ◽  
Horseshoe Crab ◽  
Proteinase Inhibitors ◽  
Binding Activity ◽  
Ester Bond ◽  
Thiol Groups ◽  
Thiol Ester ◽  
Complement Proteins ◽  
Α2 Macroglobulin

Intra-chain thiol ester bonds are present in a limited number of proteins. The thiol ester class of proteins includes vertebrate alpha 2-macroglobulin and the complement proteins C3 and C4. We report here the first instance of a thiol ester protein from an invertebrate, the alpha 2-macroglobulin proteinase-inhibitor homologue present in the plasma of the American horseshoe crab Limulus polyphemus. Our evidence is of three kinds: (1) the proteinase-binding activity of Limulus alpha 2-macroglobulin is inactivated by the low-molecular-mass primary amine methylamine; (2) the native protein is subject to autolytic fragmentation during mild thermal denaturation, yielding fragments of approx. 125 kDa and 55 kDa, whereas the methylamine-treated protein is stable under these conditions of thermal treatment; (3) new thiol groups are generated rapidly during reaction of the protein with trypsin. The demonstration of the thiol ester bond in a protein from an ancient invertebrate provides evolutionary evidence for the importance of this bond in the function of plasma forms of the alpha 2-macroglobulin-like proteinase inhibitors.

scholarly journals Sheep mast cell proteinase-1, a serine proteinase with both tryptase- and chymase-like properties, is inhibited by plasma proteinase inhibitors and is mitogenic for bovine pulmonary artery fibroblasts

1997 ◽  
Vol 323 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Alan D. PEMBERTON ◽  
Christopher M. BELHAM ◽  
John F. HUNTLEY ◽  
Robin PLEVIN ◽  
Hugh R. P. MILLER
Keyword(s):  
Mast Cell ◽  
Pulmonary Artery ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Gastrointestinal Nematode ◽  
Α2 Macroglobulin ◽  
Sds Page ◽  
Bean Trypsin Inhibitor ◽  
Β Chain

Sheep mast cell proteinase-1 (sMCP-1), a serine proteinase with dual chymase/tryptase activity, is expressed in gastrointestinal mast cells, and released systemically and on to the mucosal surface during gastrointestinal nematode infection. The potential for native plasma proteinase inhibitors to control sMCP-1 activity was investigated. Sheep α1-proteinase inhibitor (α1PI) inhibited sMCP-1 slowly, with second-order association rate constant (kass) 1.1×103 M-1·s-1, whereas sheep contrapsin inhibited trypsin (kass 2.2×106 M-1·s-1) but not sMCP-1. Western-blot analysis and gel filtration showed that when added to serum or plasma, sMCP-1 was partitioned between α1PI and α2-macroglobulin. The possibility that significant cleavage of plasma proteins could occur before sMCP-1 was inhibited was investigated using gel filtration and SDS/PAGE after adding sMCP-1 to plasma. Cleavage of ovine fibrinogen occurred in the presence of excess α1PI and α2-macroglobulin, the α-chain being cleaved C-terminally and the β-chain at the putative Lys-27. In addition, sMCP-1 was found to be mitogenic for bovine pulmonary artery fibroblasts, but was not mitogenic in the presence of soya-bean trypsin inhibitor. In terms of fibrinogen cleavage and fibroblast stimulation, sMCP-1 shows functional similarities to mast cell tryptase.

The Chemistry and Technology of Rubber-Bound and Related Novel Antioxidants

1972 ◽  
Vol 45 (1) ◽  
pp. 204-221 ◽  
Author(s):  
M. E. Cain ◽  
K. F. Gazeley ◽  
I. R. Gelling ◽  
P. M. Lewis
Keyword(s):  
Antioxidant Activity ◽  
Room Temperature ◽  
Elevated Temperatures ◽  
Protective Group ◽  
Chemically Modified ◽  
Synthetic Rubber ◽  
Naturally Occurring ◽  
Wide Range ◽  
Technological Processing ◽  
Novel Antioxidants

Abstract The discovery that C-nitrosoanilines react with olefins to give p-phenylenediamines has made possible the synthesis of powerful antioxidants from a wide range of naturally occurring and synthetic unsaturated compounds. Simple alkenes, vegetable oils, and factices may be converted to antidegradants whose behavior parallels that of commercially available materials. The reaction of 4-nitrosoanilines or 4-nitrosophenols with natural and synthetic rubber or latices gives rubber-bound antioxidants which are completely resistant to extraction by water or organic solvents. No major modification of technological processing or fabrication techniques is necessary, and the reaction takes place conveniently during vulcanization. Attachment of the protective group to the rubber molecule does not affect its antioxidant activity, but reduces its effectiveness when migration to the surface is required, e.g., in protection against ozone. Although faster at elevated temperatures, the nitroso-olefin reaction will occur at room temperature, and this allows the preparation of chemically modified raw rubbers by carrying out the reaction in latex before coagulation and drying.

Studies on Prothrombin Complex Concentrates Contact Factors, Complement Components and Proteinase Inhibitors

1984 ◽  
Vol 52 (03) ◽  
pp. 256-262 ◽  
Author(s):  
M Steinbuch ◽  
L Péjaudier ◽  
V Kichenin ◽  
M C Boffa
Keyword(s):  
Molecular Weight ◽  
Plasma Concentration ◽  
High Molecular Weight ◽  
Proteinase Inhibitors ◽  
Fold Increase ◽  
High Molecular Weight Kininogen ◽  
Prothrombin Complex Concentrates ◽  
Molecular Alteration ◽  
Complement Components ◽  
The Complement System

SummaryThe behaviour of contact factors, complement components and antiproteases during the preparation of prothrombin complex concentrates by adsorption of the clotting components on DEAE-Sephadex has been studied.The pro-enzymes: factors XII, XI and prekallikrein were removed by pre-elution in function of the salt concentration. In contrast, high molecular weight kininogen was considerably enriched in PCC preparations. C4 of the complement system displayed an analogous behaviour. Cls reached a 4-5 fold plasma concentration but C3 only 30% of the normal plasma level.The prothrombin complex concentrate contained no antithrombin III nor α2M nor α2 antiplasmin but a three fold plasma concentration of Cl-inactivator and a 15 fold increase of inter-α- trypsin inhibitor.NAPTT (Non Activated Partial Thromboplastin Time) ratios did not seem to be in accordance with either the presence or the absence of contact enzymes. Moreover 0.20 M NaCl appeared as the minimal pre-elution molarity necessary to ensure a NAPTT ratio above thrombogenic values.Molecular alteration of high molecular weight kininogen and C4 was observed and its significance discussed. Complex formation between C1-inactivator and proteases was shown to be another sign of undesirable proteolytic events.

Intramolecular general acid catalysis in the binding reactions of α2 and complement components C3 and C4

1981 ◽  
Vol 1 (6) ◽  
pp. 461-468 ◽  
Author(s):  
Stephen G. Davies ◽  
Robert B. Sim
Keyword(s):  
Acid Catalysis ◽  
Bond Cleavage ◽  
Covalent Binding ◽  
Limited Proteolysis ◽  
Peptide Bond ◽  
Complement Components ◽  
Peptide Bond Cleavage ◽  
Α2 Macroglobulin ◽  
Internal Peptide ◽  
The Complement System

The complement system proteins C3 and C4 and the plasma protease inhibitor α2-macroglobulin~ when activated by limited proteolysis, can bind covalently to other macromolecules. The three proteins also exhibit an unusual internal peptide-bond cleavage reaction when denatured. The covalent binding reaction is likely to occur by a transacylation mechanism involving an internal thioiester in the three proteins. However, the activated species of these proteins are much more reactive than simple thiolesters. Studies of molecular models of the thiolester region in C3 show that an intramolecular acid catalysis mechanism can both account for the exceptional reactivity of the activated form of these proteins and provide an explanation for the denaturation-induced peptide bond cleavage.

scholarly journals Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

2007 ◽  
Vol 407 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Ninh Doan ◽  
Peter G. W. Gettins
Keyword(s):  
Complement Protein ◽  
Complement Components ◽  
Ancestral Gene ◽  
Thiol Ester ◽  
Bait Region ◽  
Morphogenetic Protein ◽  
Complement Component C3 ◽  
Α2 Macroglobulin ◽  
Domain Interactions ◽  
Type 3

Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

Mechanical Properties and Dislocation Structure of Single Crystal Cdte Deformed in Compression at 300°C

1976 ◽  
Vol 34 ◽  
pp. 592-593
Author(s):  
Ernest L. Hall ◽  
J. B. Vander Sande
Keyword(s):  
Mechanical Properties ◽  
Single Crystal ◽  
Dislocation Structure ◽  
Room Temperature ◽  
Elevated Temperatures ◽  
Strain Curve ◽  
Optical Devices ◽  
Semiconductor Compound ◽  
Wide Range ◽  
Sample Orientation

The present paper describes research on the mechanical properties and related dislocation structure of CdTe, a II-VI semiconductor compound with a wide range of uses in electrical and optical devices. At room temperature CdTe exhibits little plasticity and at the same time relatively low strength and hardness. The mechanical behavior of CdTe was examined at elevated temperatures with the goal of understanding plastic flow in this material and eventually improving the room temperature properties. Several samples of single crystal CdTe of identical size and crystallographic orientation were deformed in compression at 300°C to various levels of total strain. A resolved shear stress vs. compressive glide strain curve (Figure la) was derived from the results of the tests and the knowledge of the sample orientation.

Spherulite Structures in a Melt Spun Fe-Ni Austenitic Steel

1985 ◽  
Vol 43 ◽  
pp. 52-53
Author(s):  
G. M. Michal ◽  
T. K. Glasgow ◽  
T. J. Moore
Keyword(s):  
Austenitic Steel ◽  
Room Temperature ◽  
Elevated Temperatures ◽  
Large Range ◽  
Amorphous Structure ◽  
Nucleation And Growth ◽  
Ni Alloys ◽  
Melt Spun ◽  
Crystal Fibers ◽  
Rapidly Solidified

Large additions of B to Fe-Ni alloys can lead to the formation of an amorphous structure, if the alloy is rapidly cooled from the liquid state to room temperature. Isothermal aging of such structures at elevated temperatures causes crystallization to occur. Commonly such crystallization pro ceeds by the nucleation and growth of spherulites which are spherical crystalline bodies of radiating crystal fibers. Spherulite features were found in the present study in a rapidly solidified alloy that was fully crysstalline as-cast. This alloy was part of a program to develop an austenitic steel for elevated temperature applications by strengthening it with TiB2. The alloy contained a relatively large percentage of B, not to induce an amorphous structure, but only as a consequence of trying to obtain a large volume fracture of TiB2 in the completely processed alloy. The observation of spherulitic features in this alloy is described herein. Utilization of the large range of useful magnifications obtainable in a modern TEM, when a suitably thinned foil is available, was a key element in this analysis.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

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