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)

Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine and phosphatidylserine (PCPS) or phosphatidylcholine alone (PC). Factor X activation was evaluated by discontinuous measurements of the amidolytic activity of the product, factor Xa, or continuously monitored using the fluorescent serine protease inhibitor 4-aminobenzamidine. The results of both techniques were verified by direct physical measurements of zymogen activation using SDS-polyacrylamide gel electrophoresis. The rate of factor X activation with PC vesicles was less than 5% of that observed with PCPS vesicles. Since factor X does not bind to vesicles containing only PC, these data suggested an important role for the substrate-membrane interaction in the catalytic cycle. The importance of the substrate-membrane interaction in the activation process was investigated by using membrane-binding proteins to compete with the substrate for combining sites on PCPS vesicles. Prothrombin fragment 1 was an inhibitor of factor X activation. The dependence of inhibition by fragment 1 on PCPS and factor X was consistent with a significant reduction in initial velocity due to the displacement of factor X from the membrane surface. The inhibition data also suggested that the membrane-bound pool of factor X was the preferred substrate for the human extrinsic Xase complex. The influence of PCPS concentrations on the rate of factor X activation was systematically investigated. Increasing concentrations of PCPS resulted in a modest change in the Km,app and a dramatic change in the Vmax,app for the reaction. The initial velocity data could be globally analyzed according to the preferential utilization of membrane-bound factor X with the intrinsic kinetic constants: Km approximately equal to 1 microM and kcat = 37 s-1 at saturating PCPS. In addition, the equilibrium parameters for the factor X-membrane interaction inferred from these studies were in excellent agreement with the directly determined values. Collectively, the data suggest that the substrate-membrane interaction must precede catalysis for the efficient activation of human factor X by the extrinsic Xase complex.
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PMID:Role of the membrane surface in the activation of human coagulation factor X. 146 22

The activation of human blood coagulation factor VII can occur by the feedback activity of either factor VIIa (autoactivation) or factor Xa. Both of these reactions are known to be enhanced by the presence of tissue factor, an integral membrane protein and the cofactor for factor VIIa. We examine here the activation of 125I-factor VII by both factor VIIa and factor Xa employing a mutant soluble form of tissue factor which has had its transmembrane and cytoplasmic domains deleted (sTF1-219). This mutant soluble tissue factor retains cofactor activity toward factor VIIa in a single-stage clotting assay but shows a strong dependence on initial plasma levels of factor VIIa (from 1 to 10,000 ng/ml) when compared to wild-type tissue factor. We show that this dependence is due to a deficiency of sTF1-219 in ability to both promote autoactivation and enhance the factor Xa-catalyzed activation of 125I-factor VII. sTF1-219 does not, however, inhibit the tissue factor-independent activation of 125I-factor VII by factor Xa. The results strongly suggest that the phospholipid anchoring region of tissue factor is essential for autoactivation and beneficial for factor Xa-catalyzed activation of 125I-factor VII. In addition, when taken together with the dependence of clotting times on initial factor VIIa levels observed with sTF1-219, these results indicate that factor VII autoactivation may be of greater importance in the initiation of blood coagulation via tissue factor than has been previously realized.
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PMID:Deletion of the membrane anchoring region of tissue factor abolishes autoactivation of factor VII but not cofactor function. Analysis of a mutant with a selective deficiency in activity. 162 32

The cytosolic domain of microsomal P450 52A3 (P450Cm1) was isolated as a soluble and functionally active protein. The NH2-terminal region that anchors the P450 protein to the endoplasmic reticulum was removed by sequence-specific proteolysis at a designed cleavage site. For that purpose, P450Cm1 was genetically engineered to establish at position 63-66 the sequence Ile-Glu-Gly-Arg, which is recognized by the restriction protease factor Xa. The modified P450 was produced in high yields as an integral membrane protein in Saccharomyces cerevisiae. In the microsomal fraction, it was accessible to factor Xa digestion, releasing a readily soluble, shortened P450 protein. For large scale preparation of the cytosolic domain, the modified P450Cm1 was first purified and then subjected to sequence-specific proteolysis. The highly purified delta(1-66)P450Cm1 exhibited unchanged spectral characteristics and catalyzed the hydroxylation of n-hexadecane with 85% of the activity determined for full-length wild-type P450Cm1. The method developed for the preparation of the cytosolic domain of P450Cm1 may be more generally applicable to facilitate structure-function studies on membrane-bound P450 forms.
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PMID:Generation of the soluble and functional cytosolic domain of microsomal cytochrome P450 52A3. 817 90

Genetic studies have shown that the expression of the pufQ gene is required for normal levels of bacteriochlorophyll biosynthesis in Rhodobacter capsulatus. Yet, the exact function of the pufQ gene is unknown, and a pufQ gene product has never been isolated. We describe the recombinant overexpression of pufQ in Escherichia coli, as well as the purification and characterization of its gene product, the 74-amino-acid PufQ protein. Site-directed mutagenesis was used to facilitate the cloning of the pufQ gene into various expression vector systems of E. coli, including pKK223-3, pLcII-FX, and pMal-c. Although high levels of pufQ transcription were evident from constructs of all three vectors, high levels of protein expression were apparent only in the pMal-c system. In vector pMal-c, the recombinant PufQ protein is expressed as a fusion with an amino-terminal maltose-binding domain. After affinity purification on an amylose column, full-length PufQ protein was released from the fusion protein by limited proteolysis with the enzyme factor Xa. The PufQ protein demonstrated a strong tendency to associate with phospholipid vesicles, consistent with the view that it is an integral membrane protein. The PufQ protein was subsequently purified by high-performance liquid chromatography and identified by amino-terminal sequence analysis. A possible role for the PufQ protein in the transport of bacteriochlorophyll biosynthetic intermediates is discussed.
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PMID:Recombinant expression of the pufQ gene of Rhodobacter capsulatus. 833 39

Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation.
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PMID:Receptor-mediated endocytosis of coagulation factor Xa requires cell surface-bound tissue factor pathway inhibitor. 862 21

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

The general amino acid permease (Gap1p) of Saccharomyces cerevisiae is an integral membrane protein that contains 12 hydrophobic regions predicted to be membrane-spanning segments. A topological reporter construct, encoding an internal 53-amino acid peptide of invertase (Suc2p) containing three Asp-X-Ser/Thr glycosylation sites, was inserted in-frame into the hydrophilic NH(2)- and COOH-terminal domains and each of the 11 hydrophilic loops that separate the 12 hydrophobic segments of Gap1p. The resulting 13 gene sandwich fusion proteins were expressed in a gap1Delta null mutant strain; 9 of these retain amino acid transport activity and are folded and correctly targeted to the plasma membrane. The glycosylation state of each of the fusion proteins was monitored; the results indicate that all 12 hydrophobic segments of Gap1p span the membrane, and the NH(2) and COOH termini are cytoplasmically oriented. These results were independently tested by isolating sealed right-side-out microsomes from sec12-1 strains expressing six different Gap1p constructs containing functional factor Xa protease cleavage sites. The pattern of factor Xa protease cleavage was found to be consistent with the presence of 12 membrane-spanning domains. Gap1p exhibited the same membrane topology in strains lacking Shr3p; therefore, Gap1p fully integrates into the ER membrane independently of this permease-specific packaging chaperone.
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PMID:A method for determining the in vivo topology of yeast polytopic membrane proteins demonstrates that Gap1p fully integrates into the membrane independently of Shr3p. 1090 20

Tissue factor (TF), the physiological trigger of the blood clotting cascade, is also the active ingredient in thromboplastin preparations which are widely used in clotting assays such as the prothrombin time (PT) test. A type I integral membrane protein, TF must be incorporated into suitable phospholipid membranes for full procoagulant activity. Several methods exist for incorporating TF into phospholipid vesicles, typically employing the formation of mixed micelles containing detergent, phospholipid and TF, followed by detergent removal or dilution below the critical micelle concentration (CMC). These methods have certain drawbacks: they may take several days to complete, employ expensive detergents, are difficult to scale up, and do not always result in complete detergent removal. In this study we have investigated the use of a variety of detergents [Triton X-100, octaethylene glycol monododecyl ether (C(12)E(8)), cholate, deoxycholate, and n-octyl-beta-D-glucopyranoside], and the use of adsorbent beads (Bio-Beads SM-2) for removing detergent, in processes to incorporate TF into proteoliposomes with high specific activity in coagulation assays. The method we have developed is rapid and readily scalable, yielding thromboplastin preparations with specific activities in plasma clotting assays that are at least as high as those made with detergent dialysis.
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PMID:Rapid and efficient incorporation of tissue factor into liposomes. 1521 99

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.
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PMID:Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI). 1574 71

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