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)

An improved method for the preparation of bovine alpha-thrombin is described. The procedure involves that activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2.200-2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strength and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of alpha-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to "aged" thrombin. In addition, the catalytic properties of alpha-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.
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PMID:On the preparation of bovine alpha-thrombin. 58 Apr 93

A comparison of the physical-chemical properties of human, bovine, and horse antithrombin III has been made. These three plasma proteins are strong inhibitors of bovine factor Xa and form a 1:1 molar complex with this coagulation enzyme. Human, bovine, and horse antithrombin III are glycoproteins containing hexose, hexosamine, and neuraminic acid. The total carbohydrate was 9, 12, and 16% for human, bovine, and horse antithrombin III, respectively. These proteins have a similar amino acid composition, although some monor variations were noted. Each antithrombin III is composed of a single polypeptide chain with an amino-terminal histidine residue. Of the first 17 amino-terminal residues, only three differences were noted between the three proteins. These occur in position 2 which is occupied by Gly, Arg, and Trp in human, bovine, and horse, respectively; position 6 which has a deletion in human antithrombin III; and position 8 where Ile in human and horse antithrombin III has been replaced by Val in the bovine preparation. The remainder of the first 17 residues is the same in all three proteins. The molecular weights for the bovine and horse preparation were 56 600 and 52 500, respectively, as determined by sedimentation equilibrium in the presence of guanidine hydrochloride. Some immunological cross-reactivity was also observed between the three different proteins.
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PMID:Characterization of human, bovine, and horse antithrombin III. 124 22

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin. 244 61

DNA sequences encoding Ile-Glu-Gly-Arg and human prorenin were joined and placed under the transcriptional control of the Escherichia coli trp promoter-operator in the expression vector pTR501. E. coli cells transformed with pTR501 expressed high levels (30% of total cell protein) of prorenin as part of a hybrid protein with the trp E gene product. The chimeric protein, accumulated in a sedimentable form, was dissolved in 6 M guanidine X HCl, purified to near homogeneity, and renatured by dialysis. The complete prorenin sequence was then excised from the renatured hybrid protein using blood coagulation factor Xa, a proteinase which is highly specific for the tetrapeptide insert Ile-Glu-Gly-Arg introduced between the 9 amino-terminal residues of the trp E gene product and the first amino acid (Thr 1) of prorenin. Human prorenin thus obtained was readily activatable with trypsin and showed close similarities to naturally occurring prorenin in its biochemical and immunochemical properties.
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PMID:Synthesis and characterization of human prorenin in Escherichia coli. 353 94

We have investigated the in vitro reconstitution of the 24-meric inner core domain (E2c) of the transacylase (E2) component of bovine branched-chain alpha-keto acid dehydrogenase complex. The yield of recombinant E2c (amino acid residues 161-421 of bovine E2) expressed in Escherichia coli was markedly increased by fusing the bacterial maltose-binding protein (MBP) to the amino terminus of bovine E2c. Following factor Xa digestion to remove the MBP moiety, E2c was completely unfolded in 4.5 M guanidine HCl (Gdn.HCl). The denatured E2c monomers (apparent M(r) = 27,000) were diluted 100-fold at 25 degrees C into a refolding buffer containing 5 mM Mg-ATP and a 4-fold molar excess of chaperonins GroEL and GroES. Full E2 activity was recovered in 45 min. Omission of the chaperonins in the refolding buffer failed to recover any E2 activity. Recovery of E2 activity obeyed hyperbolic kinetics as a function of the chaperonin-to-E2c molar ratio and showed a requirement for hydrolysis of Mg-ATP. A stable GroEL-E2c complex was isolated which, in the presence of GroES and Mg-ATP, generated active E2c 24-mers. Dissociation of recombinant E2c 24-mers into active trimers was achieved by incubation in 1.5 M Gdn.HCl at 25 degrees C. The E2c trimers with an apparent M(r) of 84,000 were isolated by sucrose density gradient centrifugation in the presence of the chaotropic reagent. Removal of 1.5 M Gdn.HCl resulted in the spontaneous reassembly of trimers into the native 24-mer structure independent of chaperonins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro reconstitution of the 24-meric E2 inner core of bovine mitochondrial branched-chain alpha-keto acid dehydrogenase complex: requirement for chaperonins GroEL and GroES. 791 32

A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.
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PMID:Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli. 822 27

The in vivo synthesis of many target proteins or polypeptides has been enhanced dramatically and their purification facilitated through the use of gene fusion techniques which lead to the expression of fusion proteins. This approach was used to characterize the product formed in Escherichia coli encoded by a DNA construct comprising malE, the gene encoding maltose binding protein, linked to a small 30 nucleotide region which, in turn, was linked to pyrB, the gene encoding the catalytic (c) chains of aspartate transcarbamoylase (ATCase). The resulting fusion protein, MBP-C, was produced in excellent yield and readily purified in two steps because of its binding to an amylose column and displacement by maltose. The complex was studied by both sedimentation velocity and sedimentation equilibrium and shown to be a trimer of c chains with one MBP linked covalently to each chain. Treatment of the fusion protein with factor Xa cleaved each chain at the tetrapeptide encoded by the linker region yielding purified MBP with a minor modification at the C-terminus and the catalytic (C) trimer of ATCase. The MBP-C complex was fully active as an enzyme and could be reversibly denatured in 6 M urea. Scanning calorimetry studies on the fusion protein demonstrated that the MBP domain melted at the same temperature as did the purified protein. Similarly, the Tm for the C trimer in the complex was identical to the value for C trimer isolated from ATCase. Moreover, the thermal stability of the C trimer in the MBP-C complex was greatly enhanced by the addition of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), just as observed with purified C trimer. Analogous denaturation experiments with varying concentrations of guanidine-HCl indicated that the fusion protein was denatured at much lower concentration of denaturant than observed for C trimer. These experiments demonstrate that the linker between the two structural genes encodes a polypeptide of sufficient length to permit independent folding and assembly of each protein and permit the subsequent specific cleavage at the factor Xa recognition site, thereby yielding both active proteins.
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PMID:A bifunctional fusion protein containing the maltose-binding polypeptide and the catalytic chain of aspartate transcarbamoylase: assembly, oligomers, and domains. 867 17

Decorin is a small leucine-rich proteoglycan which is a component of the extracellular matrix of many connective tissues. We have developed a method for expression and purification of decorin as a fusion protein in Escherichia coli. A PCR product coding for the 330-amino-acid-residue secreted form of bovine decorin core protein was cloned into the vector pMal-c for expression in E. coli as a maltose-binding protein (MBP) fusion. Expressed MBP-decorin tended to form insoluble aggregates resistant to degradation by E. coli intracellular proteases. Fusion protein was therefore solubilized from bacterial inclusion bodies using guanidine HCl and refolded by dilution of the chaotrope to minimally denaturing conditions and disulfide shuffling. Final purification included amylose resin affinity chromatography and size exclusion chromatography. The 79-kDa MBP-decorin fusion protein could be completely cleaved by factor Xa protease in 24 h to yield 43-kDa MBP and 36-kDa decorin core protein. The decorin core protein was separated from MBP and factor Xa protease by DEAE-Sephacel chromatography. Using a solid-phase assay, we have characterized its binding affinity for extracellular matrix components known to interact with tissue-derived decorin including collagen type VI, collagen type I, and fibronectin. The purification and refolding protocol described here may be generally applicable to bacterial expression of other members of this growing family of related small leucine-rich proteoglycan core proteins.
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PMID:Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion. 881 84

A truncated variant of the hepatitis B virus X gene (HBx) was cloned into the fusion expression vector of pGEX-3X (Pharmacia), resulting in a GST-HBx fusion gene construction (pGEX-3XXBF). This plasmid was transformed into and expressed by the Escherichia coli strain DH5. More than 80% of the expressed fusion protein was found in the insoluble fraction (inclusion body) of the cell lysate. The fusion protein was selectively extracted from the inclusion bodies with 8 M urea at pH 6.5, and it was refolded by diluting 3-fold with deionized distilled water at 4 degrees C. The in vitro cleavage of the refolded fusion protein by factor Xa at about 2-3 mg ml-1 in the presence of 2.66 M urea at pH 6.5 was complete. The final steps of purification involved precipitation of the cleaved proteins with ammonium sulphate, solubilization in guanidine hydrochloride and separation on a Superdex 75 FPLC column. With this approach, following an inclusion body strategy and a beneficial in vitro refolding, a predominantly hydrophobic and highly disulphide-bonded protein was produced in preparative scale for subsequent diagnostic use.
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PMID:An alternative purification protocol for producing hepatitis B virus X antigen on a preparative scale in Escherichia coli. 930 71

Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.
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PMID:Monoclonal antibody light chain with prothrombinase activity. 1082 60


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