Browse
Submit Data
Databases
API
Help

Dataset Information

5 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Preparation of anhydrothrombin and characterization of its interaction with natural thrombin substrates.

ABSTRACT: Thrombin is a serine proteinase that plays a key role in thrombosis and haemostasis through its interaction with several coagulation factors. Anhydrothrombin was prepared from PMSF-inactivated thrombin under alkaline conditions, and the folded anhydrothrombin was successfully recovered after dialysis in the presence of glycerol. Anhydro-derivatives of factor Xa, factor VIIa and activated protein C could also be prepared essentially by the same procedure. Anhydrothrombin retained affinity for various natural substrates of thrombin, including fibrinogen, factor VIII, factor XIII and protein C. In addition, these proteins were bound to anhydrothrombin-agarose in a reversible manner. The K(d) values for factor VIII, fibrinogen, factor XIII and protein C were 1.2x10(-8), 4.4x10(-8), 2.8x10(-7) and 8.1x10(-5) M, respectively. Thus thrombin substrates known to interact with the exosite I of thrombin demonstrated high affinity for anhydrothrombin. Furthermore, in the presence of Na+, substantial enhancement of the association rate constant (k(ass)) was observed for interactions of fibrinogen and factor VIII with anhydrothrombin. These results suggest that anhydrothrombin is useful in the purification of thrombin substrate proteins as well as in the investigation of detailed interactions between thrombin and these substrates in their activation or degradation processes.

SUBMITTER: Hosokawa K

PROVIDER: S-EPMC1221657 | biostudies-other | 2001 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Preparation and Characterization of UV-Curable Acrylic Membranes Embedding Natural Antioxidants.

Project description:We examine the behaviour of acrylic resin-based membranes containing natural anti-oxidants, such as GallaChinensis tea powder extract (TP) and Taiwanese green propolis (TGP), in different concentrations ranging between 5 and 20 wt %. Membrane morphology was investigated by means of Environmental Scanning Electron Microscopy (ESEM), while the UV-curing reaction was monitored by Fourier-Transform Infra-red (FTIR) spectroscopy. In most cases Thermogravimetric (TG), Differential Scanning Calorimetric (DSC) and Dynamo-mechanical Thermal (DMT) analyses showed that the desirable characteristics of the UV-cured acrylic resin are not substantially altered by the presence of the organic fillers. The release kinetics of polyphenols and flavonoids, determined in water for TP-containing membranes (ETx) and in ethanol/water mixture (7:3 v/v) for TGP-containing ones (EPx), was satisfactory, reaching a plateau after 24 h. Finally, preliminary antibacterial tests against S. Epidermidis were performed on the membranes with higher additive amount and gave positive results for ET-type; on the contrary, no inhibitory effect was observed for the tested EP-type membranes.

| S-EPMC7077452 | biostudies-literature

Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors.

Project description:Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.

| S-EPMC1146971 | biostudies-other

Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45).

Project description:The specificity of insulin proteinase (EC 3.4.99.45) has been difficult to categorize using only its natural substrates. By exploiting the fact that two substrates competing for the same enzyme inhibit one another, we have found some new substrates of the insulin proteinase from porcine muscle. Two of these substrates, a tryptic fragment of BSA and a fragment of cytochrome c, have been shown to be cleaved at a single site. The albumin fragment, as well as another fragment of cytochrome c., have susceptibilities (Vmax/Km) comparable with that of insulin. In a second aspect of the study, the porcine-muscle enzyme was shown to be related to other members of its superfamily in that it was immunoprecipitated by a monoclonal antibody raised against the insulin-degrading enzyme from human red blood cells and has the same cleavage sites on insulin as has the rat skeletal-muscle insulin proteinase. We note, however, a possible discrepancy between our results and those of another group regarding the subunit size (110 kDa) of the immunoprecipitated material.

| S-EPMC1137316 | biostudies-other

Interaction of thrombin with sucrose octasulfate.

Project description:The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombin's activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 ?M, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombin's catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.

| S-EPMC3172994 | biostudies-literature

Interaction of thrombin with PAR1 and PAR4 at the thrombin cleavage site.

Project description:Investigations determined the critical amino acids for alpha-thrombin's interaction with protease-activated receptors 1 and 4 (PAR1 and PAR4, respectively) at the thrombin cleavage site. Recombinant PAR1 wild-type (wt) exodomain was cleaved by alpha-thrombin with a Km of 28 microM, a kcat of 340 s-1, and a kcat/Km of 1.2 x 10(7). When the P4 or P2 position was mutated to alanine, PAR1-L38A or PAR1-P40A, respectively, the Km was unchanged, 29 or 23 microM, respectively; however, the kcat and kcat/Km were reduced in each case. In contrast, when Asp39 at P3 was mutated to alanine, PAR1-D39A, Km and kcat were both reduced approximately 3-fold, making the kcat/Km the same as that of PAR1-wt exodomain. Recombinant PAR4-wt exodomain was cleaved by alpha-thrombin with a Km of 61 microM, a kcat of 17 s-1, and a kcat/Km of 2.8 x 10(5). When the P5 or P4 position was mutated to alanine, PAR4-L43A or PAR4-P44A, respectively, there was no change in the Km (69 or 56 microM, respectively); however, the kcat was lowered in each case (9.7 or 7.7 s-1, respectively). Mutation of the P2 position (PAR4-P46A) also had no effect on the Km but markedly lowered the kcat and kcat/Km approximately 35-fold. PAR1-wt exodomain and P4 and P3 mutants were noncompetitive inhibitors of alpha-thrombin hydrolyzing Sar-Pro-Arg-pNA. However, PAR1-P40A displayed a mixed type of inhibition. Mutation of P4, P3, or P2 had no effect on the Ki. All PAR4 exodomains were competitive inhibitors of alpha-thrombin. Mutation of P5, P4, or P2 had no effect on the Ki. These investigations show that Leu at P4 in PAR1 or P5 in PAR4 critically influences the kinetics of alpha-thrombin binding and cleavage of PAR1 and PAR4 exodomains. It also implies that factors other than the hirudin-like binding region on PAR1 exodomain predominate in influencing PAR1 cleavage on cells.

| S-EPMC2553362 | biostudies-literature

Characterization of the nonheme iron center of cysteamine dioxygenase and its interaction with substrates.

Project description:Cysteamine dioxygenase (ADO) has been reported to exhibit two distinct biological functions with a nonheme iron center. It catalyzes oxidation of both cysteamine in sulfur metabolism and N-terminal cysteine-containing proteins or peptides, such as regulator of G protein signaling 5 (RGS5). It thereby preserves oxygen homeostasis in a variety of physiological processes. However, little is known about its catalytic center and how it interacts with these two types of primary substrates in addition to O2 Here, using electron paramagnetic resonance (EPR), Mössbauer, and UV-visible spectroscopies, we explored the binding mode of cysteamine and RGS5 to human and mouse ADO proteins in their physiologically relevant ferrous form. This characterization revealed that in the presence of nitric oxide as a spin probe and oxygen surrogate, both the small molecule and the peptide substrates coordinate the iron center with their free thiols in a monodentate binding mode, in sharp contrast to binding behaviors observed in other thiol dioxygenases. We observed a substrate-bound B-type dinitrosyl iron center complex in ADO, suggesting the possibility of dioxygen binding to the iron ion in a side-on mode. Moreover, we observed substrate-mediated reduction of the iron center from ferric to the ferrous oxidation state. Subsequent MS analysis indicated corresponding disulfide formation of the substrates, suggesting that the presence of the substrate could reactivate ADO to defend against oxidative stress. The findings of this work contribute to the understanding of the substrate interaction in ADO and fill a gap in our knowledge of the substrate specificity of thiol dioxygenases.

| S-EPMC7450127 | biostudies-literature

Ni(II) Complexes with Schiff Base Ligands: Preparation, Characterization, DNA/Protein Interaction and Cytotoxicity Studies.

Project description:In this study, two Ni(II) complexes, namely [Ni(HL1)?(OAc)?] (1) and [Ni(L2)?] (2) (where HL1 and HL2 are (E)-1-((1-(2-hydroxyethyl)-1H-pyrazol-5-ylimino)methyl)-naphthalen-2-ol) and (E)-ethyl-5-((2-hydroxynaphthalen-1-yl)methyleneamino)-1-methyl-1H-pyrazole-4-carboxylate, respectively), were synthesized and characterized by X-ray crystallography, Electrospray Ionization Mass Spectrometry (ESI-MS), elemental analysis, and IR. Their uptake in biological macromolecules and cancer cells were preliminarily investigated through electronic absorption (UV-Vis), circular dichroism (CD) and fluorescence quenching measurements. Bovine serum albumin (BSA) interaction experiments were investigated by spectroscopy which showed that the complexes and ligands could quench the intrinsic fluorescence of BSA through an obvious static quenching process. The spectroscopic studies indicated that these complexes could bind to DNA via groove, non-covalent, and electrostatic interactions. Furthermore, in vitro methyl thiazolyl tetrazolium (MTT) assays and Annexin V/PI flow cytometry experiments were performed to assess the antitumor capacity of the complexes against eight cell lines. The results show that both of the complexes possess reasonable cytotoxicities.

| S-EPMC6151616 | biostudies-literature

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

Project description:The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process.In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale.Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters.An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces.These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions.

| S-EPMC4398634 | biostudies-literature

Flexibility of the thrombin-activatable fibrinolysis inhibitor pro-domain enables productive binding of protein substrates.

Project description:We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

| S-EPMC2992258 | biostudies-literature

Preparation of Monodisperse Giant Unilamellar Anchored Vesicles Using Micropatterned Hydrogel Substrates.

Project description:Giant unilamellar vesicles (GUVs) are model membrane systems consisting of a single lipid bilayer separating an inner lumen from the outer solution, with dimensions comparable to that of eukaryotic cells. The importance of these biomimetic systems has recently grown with the development of easy and safe methods to assemble GUVs from complex biorelevant compositions. However, size and position control is still a key challenge for GUV formation and manipulation. Here, a gel-assisted formation method is introduced, able to produce arrays of giant unilamellar anchored vesicles (GUAVs) with a predetermined narrow size distribution. The approach based on micropatterned gel substrates of cross-linked poly(N-isopropylacrylamide) allows performing parallel measurements on thousands of immobile unilamellar vesicles. Such power and flexibility will respond to the growing need for developing platforms of biomimetic constructs from cell-sized single bilayers.

| S-EPMC6648857 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data