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)

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.
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PMID:An activator of blood coagulation factor X from the venom of Bungarus fasciatus. 859 79

We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli. Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge. PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg). E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture. This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci. Recombinant PHM was not distinguishable from natural PHM in all aspects observed.
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PMID:Improved production of phenomycin by a genetically engineered Escherichia coli. 860 92

We have previously showed that factor X activator of Russell's viper venom (RVV-X) contains six N-linked oligosaccharide chains: four in the heavy chain and one in each of the two light chains [Gowda, D.C., Jackson, C.M., Hensley, P., & Davidson, E.A. (1994) J. Biol. Chem. 269, 10644-10650]. In the present study, we have investigated the role of the carbohydrate moieties in the structure and functional activity of RVV-X. Sequential removal of sugar residues from the terminal ends by exoglycosidases, up to 50% of total carbohydrates, did not significantly alter the activity of RVV-X, demonstrating that the peripheral carbohydrate moieties are not involved in interactions with factor X. However, removal of whole oligosaccharide chains by N-glycanase caused an almost total loss of the ability of RVV-X to activate factor X to factor Xa. In parallel with these observations, circular dichroism spectroscopy showed that complete deglycosylation, but not the removal of peripheral sugars, caused a significant change in the secondary structure. Together, these data demonstrate that the oligosaccharide chains are necessary for the functional activity, and that the trimannosylchitobiose core residues are sufficient for the maintenance of the native polypeptide structure.
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PMID:Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure. 863 44

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

Recombinant vasoactive intestinal polypeptide (VIP) analogs were expressed in Escherichia coli as a fusion protein containing tandemly repeated multiple copies of a synthetic VIP gene joined to glutathione S-transferase. The encoded protein contains VIP units separated by a linker peptide, potentially excisable by a double cleavage with endoprotease factor Xa and hydroxylamine. Expression of different polyVIP genes, from 1 to 32 units, was detected and the production of a 16 VIP polymer was performed. MonoVIP analogs appended by 5 or 10 amino acids at their C terminus were released by factor Xa from this polymerized product. They were then submitted to hydroxylamine cleavage to remove the linker sequence to finally obtain a recombinant VIP analog devoid of any amino acid extension. The biological activity of the recombinant polyVIP and VIP analogs was tested. Although less efficient than the natural neuropeptide, some of these components bound to VIP receptor, activated adenylate cyclase in human colonic adenocarcinoma cells and displayed a relaxation activity on guinea pig tracheal rings.
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PMID:Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs. 872 6

Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum. As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites. Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity. Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis. The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains.
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PMID:Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex. 881 Mar 33

Human group II secretory phospholipase A2 (sPLA2) is an enzyme found in the alpha granules of platelets and at inflammatory sites. Although its physiological function is unclear, sPLA2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA2 activities, we developed a total chemical synthesis of sPLA2 by chemical ligation of large unprotected peptides. The synthetic segments PLA2-(1-58)-alphaCOSCH2COOH and PLA2-(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly58 and Cys59. The 124-residue polypeptide product (mass: 13,920 +/- 2 Da) was folded to yield one major product (mass: 13,905 +/- 1 Da), the loss of 15 +/- 3 Da reflecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA2 showed alpha-helix, beta-structure, and random coil contents consistent with those found in the crystal structure of sPLA2. Synthetic sPLA2 had kcat and Km values identical to those of recombinant sPLA2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA2, like recombinant sPLA2, inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca2+, and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca2+. Thus, synthetic sPLA2 is a phospholipid-independent anticoagulant like recombinant or natural sPLA2. This study demonstrates that chemical synthesis of sPLA2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA2 analogs for structure-activity studies of anticoagulant, lipolytic, or inflammatory activities.
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PMID:Total chemical synthesis of enzymatically active human type II secretory phospholipase A2. 922 75

An expression vector, pOVEX, has been designed and constructed, combining the advantages of the expression vectors pGEX-3X and pJC2o. The pOVEX vector produces a fusion protein with the 24 kD Onchocerca volvulus glutathione S-transferase (OvGST2) which is easy to purify in one step from bacterial extracts under non-denaturing conditions using glutathione-sepharose chromatography. High yields of fusion protein were produced from this T7 RNA polymerase-dependent expression vector, which were then cleaved by digestion with the factor Xa protease to separate the OVGST2 polypeptide from the expressed protein of interest. This vector will be particularly useful to O. volvulus investigators for the production of O. volvulus antigens for the analyses of host humoral and cellular responses to these proteins and for immunization studies.
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PMID:pOVEX vector: prokaryotic expression and purification of onchocerciasis vaccine candidate antigens as fusion proteins with the 24 kD Onchocerca volvulus glutathione S-transferase. 927 Jul 37

A cDNA encoding a short polypeptide blocker of K+ channels, kaliotoxin 2 (KTX2), from the venom of the North African scorpion Androctonus australis was expressed in the periplasmic space of Escherichia coli. KTX2 was produced as a fusion protein with the maltose binding protein followed by the recognition site for factor Xa or enterokinase preceding the first amino acid residue of the toxin. The fully refolded recombinant KTX2 (rKTX2) was obtained (0.15-0.30 mg/l of culture) and was indistinguishable from the native toxin according to chemical and biological criteria. An N-extended analogue of KTX2 exhibiting three additional residues was also expressed. This analogue had 1000-fold less affinity for the 125I-kaliotoxin binding site on rat brain synaptosomes than KTX2. Conformational models of KTX2 and its mutant were designed by amino acid replacement using the structure of agitoxin 2 from Leiurus quinquestriatus as template, to try to understand the decrease in affinity for the receptor.
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PMID:Influence of a NH2-terminal extension on the activity of KTX2, a K+ channel blocker purified from Androctonus australis scorpion venom. 939 89

We previously reported that retroviral vectors displaying epidermal growth factor (EGF) as part of a chimeric envelope glycoprotein are sequestered upon binding to EGF receptor (EGFR)-positive target cells, leading to loss of infectivity. In the current study, we have displayed stem cell factor (SCF) on beta-galactosidase-transducing ecotropic and amphotropic retroviral vector particles as a factor Xa protease-cleavable N-terminal extension of the envelope glycoprotein. Viral incorporation of the SCF chimeric envelopes was demonstrated by immunoblotting of pelleted virions and their specific attachment to Kit receptors was demonstrated by flow cytometry. Gene transfer studies showed that when SCF was displayed on an amphotropic envelope, the infectivity of the SCF-displaying vectors was selectively inhibited on Kit-expressing cells, but could be restored by adding soluble SCF to block the Kit receptors or by cleaving the displayed SCF domain from the vector particles with factor Xa protease. The host range properties of EGF-displaying and SCF-displaying vectors were then compared in cell mixing experiments. When EGFR-positive cancer cells and Kit-positive hematopoietic cells were mixed and exposed to the different engineered vector particles, the cancer cells were selectively transduced by the SCF-displaying vector and the hematopoietic cells were selectively transduced by the EGF-displaying vector. Retroviral display of polypeptide growth factors can therefore provide the basis for a novel inverse targeting strategy with potential use for selective transduction of hematopoietic or nonhematopoietic cells (eg, cancer cells) in a mixed cell population.
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PMID:Inverse targeting of retroviral vectors: selective gene transfer in a mixed population of hematopoietic and nonhematopoietic cells. 947 49


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