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)

To probe the functional role of tryptophan 49 in human antithrombin III, a mutant antithrombin, W49K, has been expressed in baby hamster kidney cells. The mutation reduces the affinity for heparin pentasaccharide by 1.8 kcal mol-1 but does not alter the heparin enhancement of the rate of factor Xa inhibition. 1H NMR spectra of W49K antithrombin show that the structure of the protein and the mode of heparin binding appear to be unaltered by the mutation, although tryptophan 49 is perturbed by heparin binding. 19F NMR spectra of 6-fluorotryptophan-substituted antithrombin show that tryptophan 49 is in a solvent-exposed environment. The heparin-induced fluorescence enhancement of W49K antithrombin is significantly different from that of wild-type antithrombin. Pentasaccharide induces only a 24% enhancement of antithrombin fluorescence, while high affinity heparin induces an enhancement of 40%. The results indicate that tryptophan 49 is probably a heparin contact residue but can be mutated without altering the remaining heparin-antithrombin interactions or the heparin-induced conformational change and resultant activation toward Factor Xa. Hydrophobic as well as charge interactions are thus probably involved in the specificity of the antithrombin-heparin pentasaccharide interaction. The lower fluorescence enhancements suggest that the heparin-induced 40% fluorescence enhancement used as the hallmark of activating heparin species is not the best indicator of the structural change in antithrombin that results in enhancement of the rate of proteinase inhibition.
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PMID:Role of tryptophan 49 in the heparin cofactor activity of human antithrombin III. 140 May 4

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond.
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PMID:Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397. 190 72

According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.
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PMID:Influence of chemical modification of tryptophan residues on the properties of human antithrombin III. 231 91

Human prothrombin and prothrombin fragment 1 were demonstrated to bind to Phenyl-TSK columns in the presence of 5.0 mM calcium ions but not in the presence of either magnesium ions or manganese ions. The calcium-dependent interaction of prothrombin fragment 1 is markedly reduced upon oxidation of approximately one mole of tryptophan per mole of protein. The ability of prothrombin fragment 1 to inhibit prothrombin activation by factor Xa in the presence of calcium ions and phospholipid is also markedly reduced by reaction with N-bromosuccinimide. These results provide the first demonstration of a calcium-specific site in prothrombin outside of the "GLA domain".
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PMID:A hydrophobic site in human prothrombin present in a calcium-stabilized conformer. 319 39

Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of 125I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of 125I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of 125I-AT III from the vessel segment. Dissociation of 125I-AT III from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-125I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of 125I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on 125I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.
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PMID:Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity. 396 7

We have utilized circular dichroism spectroscopy to examine the interaction of antithrombin with heparin-derived oligosaccharides and mucopolysaccharides of various sizes. Our studies demonstrate that the various complexes exhibit two major types of chiral absorption spectra. The first of these patterns is seen when octasaccharide, decasaccharide, dodecasaccharide, or tetradecasaccharide fragments bind to the protease inhibitor. The circular dichroism spectra of these complexes when compared to the spectrum of free antithrombin show several distinguishing characteristics. On the one hand, there is a marked general increase in positive chiral absorption that is maximal at 296 and 288 nm and 290 and 282.5 nm. These observations indicate perturbation of "buried" and "exposed" tryptophan residues. On the other hand, a significant augmentation in circular dichroism that peaks at 269.5 and 263 nm is noted. These findings are probably due to the summed positive and negative contributions arising from tryptophan residue(s), disulfide bridge(s), and phenylalanine residue(s). Given that these heparin fragments are able to accelerate factor Xa-antithrombin interactions but not thrombin-antithrombin interactions, the above spectral transitions must be associated with either the binding of a critical domain of the oligosaccharides to the protease inhibitor or the "activation" of the protease inhibitor with respect to factor Xa neutralization. The second of these patterns is apparent when octadecasaccharide, low molecular weight heparin (6,500), and high molecular weight heparin (22,000) interact with antithrombin. The circular dichroism spectra of these complexes compared to the spectrum of free protease inhibitor are similar to the first pattern except for changes within the 292- to 282-nm and 275- to 255-nm regions. The subtraction of the first pattern from the second pattern reveals a shallow negative band between 300 and 275 nm with potential negative minima at 290 and 283 nm as well as a deep negative band between 275 and 255 nm with possible negative minima at 268 and 262 nm. This chiral absorption profile is most likely to arise from conformational changes of a disulfide bridge(s). However, we cannot completely exclude the possibility that the above circular dichroism difference curve might be explained on the basis of transitions originating from a tryptophan residue(s). Given our method for generating the above data, these spectral alterations must be associated with the binding of a second critical domain of the mucopolysaccharide to antithrombin that is required for rapid complex formation with thrombin or the activation of the protease inhibitor with respect to the neutralization of the latter enzyme.
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PMID:Circular dichroism spectroscopy of heparin-antithrombin interactions. 696 2

Human phenylalanine hydroxylase (hPAH) contains three tryptophan residues (W120, W187, and W326). All three tryptophan residues were mutated to phenylalanine either as single mutants or in combination, and one tryptophan was also mutated to isoleucine. The mutant enzymes were expressed in Escherichia coli and purified as fusion proteins with maltose-binding protein and a linker region containing a recognition site for the serine protease factor Xa. After cleavage by factor Xa, all mutants were purified to homogeneity, and the kinetic and spectroscopic properties of the proteins were studied. All the proteins had high catalytic activities, but the affinity for phenylalanine was increased for the W1201 and W120F mutants, and decreased for the W187F and W326F mutants. Using steady-state fluorescence spectroscopy, the contributions of the individual tryptophan residues to the total intrinsic fluorescence of the protein were estimated. On the basis of measurements of mutants containing only one tryptophan, it was calculated that W120, W187, and W326 account for approximately 61, 13, and 26% of the total tryptophan fluorescence of hPAH, respectively, while the positions of the emission maxima (335.5-336.5 nm) and the widths at half-height (55-60 nm) of the emission spectra of the individual tryptophans were rather similar. After incubation with phenylalanine, the quantum yield of wild-type hPAH increases by 15%, and the emission maximum is shifted from 336.5 to 347 nm. This effect is mainly due to changes in the W120 emission. On the basis of fluorescence quenching studies, this amino acid is the most surface-exposed of the tryptophan residues.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli. 754 12

Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Both techniques suggest that rTAP is rich in beta-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to fluorescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine residues exhibit greater solvent exposure upon reduction of the cystines as indicated by fluorescence spectra and second derivative UV spectroscopy. A considerable amount of beta-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced peptide binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of the reduced peptide at pH 2 is, however, similar to that at pH 7 as judged by CD spectroscopy. The reduced form of rTAP at acidic pH thus resembles a molten globule-like state.
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PMID:Spectroscopic characterization of tick anticoagulant peptide. 776 77

A pMAL-vector-based plasmid clone with synthetic tac-promotor effectively expressing full-length terminal repeats protein 1 (TP1) of Epstein-Barr virus (EBV) in E. coli was constructed. It is important that the N-terminal region of recombinant TP1 was represented by a maltose-binding protein. The latter can be used to separate TP1 from bacterial lysate by affinity chromatography. Moreover, after treatment with the proteolytic factor Xa, full-length TP1 can be recovered in a discrete form. On the basis of the pATH tryptophan-regulated vector, several plasmid clones expressing different fragments of N- and C-terminal regions of TP1 were also constructed. This collection of recombinant proteins could be used as an important tool for obtaining corresponding antisera and for immunological mapping of the TP1 molecule.
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PMID:[Expression of the Epstein-Barr virus terminal repeat protein 1 in Escherichia coli cells]. 799 Aug 31

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
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PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13


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