Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.
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PMID:Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin. 886 82

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.
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PMID:Sequence of a new Bowman-Birk inhibitor from Torresea acreana seeds and comparison with Torresea cearensis trypsin inhibitor (TcTI2). 889 2

The binding of heparin to antithrombin greatly accelerates the rate of inhibition of the target proteinases thrombin and factor Xa. Acceleration of the rate of inhibition of factor Xa involves a conformational change in antithrombin that is translated from the heparin binding site to the reactive center loop. A mechanism has been proposed for generation and propagation of the conformational change in which the binding of the negatively charged heparin reduces ionic repulsions between positively charged residues on and adjacent to the D-helix in the heparin binding site of antithrombin (van Boeckel, C. A. A., Grootenhuis, P. D. J., and Visser, A. (1994) Nature Struct. Biol. 1, 423-425). This charge neutralization is proposed to elongate the D-helix and initiate the conformational change which is then translated to the reactive center loop. Several basic residues, including arginine 132 and lysine 133, were predicted to be important both in heparin binding and in this mechanism of heparin activation. To test both the helix extension mechanism and the role of these two residues in heparin binding and factor Xa inhibition, we individually changed arginine 132 and lysine 133 to uncharged methionine by site-directed mutagenesis. The Kd values for binding of R132M and K133M variants to the high affinity pentasaccharide were weakened only 2.3- and 4.5-fold respectively, suggesting a location for R132 and K133 peripheral to the main pentasaccharide binding site. However, the Kd values for long chain high affinity heparin were weakened at least 17-fold for both R132M and K133M, indicating involvement of each residue in binding extended chain heparin species. These reductions in affinity were ionic strength-dependent. The rates of inhibition of factor Xa and thrombin by each variant, however, were indistinguishable from those of control antithrombin, and the accelerations of the rate of inhibition produced by heparin were normal. We conclude that neither arginine 132 nor lysine 133 plays an important role in the binding of heparin pentasaccharide or in the mechanism of heparin activation, suggesting that D-helix extension through charge neutralization is not the mechanism for transmission of conformational change from the heparin binding site to the reactive center region. Arginine 132 and lysine 133 do, however, play a role in tight binding of longer chain heparin species through ionic interactions.
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PMID:Role of arginine 132 and lysine 133 in heparin binding to and activation of antithrombin. 891 May 98

Plasminogen has been immobilized onto a segmented polyurethane containing amino groups, using glutaraldehyde as coupling agent. It was also aspecifically adsorbed, for sake of comparison, onto polyurethane films containing different functional groups and, in particular, epsilon-amino caproic acid and lysine residues. The differently immobilized plasminogen has been converted to plasmin by activation with urokinase, and the percentage of active plasmin for the various polymer films was determined using a tripeptide (S-2251) as a synthetic substrate. The biological behaviour of the differently treated polymer films has been evaluated in vitro by measurements of partial thromboplastin time (PTT) and platelet adhesion.
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PMID:Preparation and evaluation of polyurethane surfaces containing immobilized plasminogen. 904 Oct 39

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.
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PMID:Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78. 905 3

Recombinant native antithrombin III (ATIII) and two genetic variants with glutamine substitutions at lysine residues 114 and 139 were expressed in insect cells using a baculovirus-driven expression system. The purified proteins were used to evaluate the potential role(s) of these residues in the pentasaccharide-mediated activation of ATIII. The second order rate constants for the inhibition of factor Xa by both of the genetic variants were nearly identical to those of recombinant native ATIII, indicating that the glutamine substitutions did not result in serious protein conformational changes. The glutamine substitution at lysine 139 had no effect on the pentasaccharide-mediated activation of ATIII toward factor Xa. In contrast, lysine 114 was found to be critical in the activation of ATIII toward factor Xa. No activation was observed, even at a pentasaccharide concentration 10 times higher than that required to activate recombinant native ATIII. These data are the first to demonstrate a pivotal role for lysine 114 in the pentasaccharide-mediated activation of ATIII.
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PMID:Lysine residue 114 in human antithrombin III is required for heparin pentasaccharide-mediated activation. 906 21

Protein S is a vitamin K-dependent plasma protein that acts as a cofactor of activated protein C (APC) and as an inhibitor of the prothrombinase complex. We previously reported that protein S-Tokushima, found in a patient with heterozygous protein S deficiency and severe venous thrombotic disease, has a substitution of Glu for Lys-155 in the second epidermal growth factor (EGF)-like domain of protein S. In the present study, we characterized the role of protein S, which is required for the expression of the APC cofactor activity and for the inhibition of the prothrombinase complex activity, using recombinant normal protein S (rPS-N) and protein S-Tokushima (rPS-T), expressed in BHK cells. rPS-N as well as plasma protein S showed APC cofactor activity, but rPS-T did not. Both rPS-N and rPS-T equally bound to phospholipids (Cephalin) and C4b-binding protein fixed to microwells. APC bound to rPS-N and plasma protein S, but did not bind to rPS-T. Although rPS-N as well as plasma protein S inhibited the platelet prothrombinase complex activity, rPS-T did not. Factor Xa bound to rPS-N but not to rPST. Binding of rPS-N to biotinylated factor Va in solution phase did not differ significantly from that of rPS-T. The dysfunction of protein S-Tokushima is suggested to be mainly caused by a lack of interaction with APC required for the expression of the APC cofactor activity and by a lack of interaction with factor Xa required for the inhibition of the prothrombinase complex activity.
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PMID:Characterization of dysfunctional protein S-Tokushima (K155-->E) in relation to the molecular interactions required for the regulation of blood coagulation. 911 55

One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade. We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis. Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa). The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa. hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243). hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma. The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma. Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA. Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time. These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease.
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PMID:A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent. 912 26

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.
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PMID:Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin. 915 Feb 44

A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.
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PMID:Purification and primary structure determination of a Bowman-Birk trypsin inhibitor from Torresea cearensis seeds. 916 81


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