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)

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.
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PMID:Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. 1137 81

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.
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PMID:SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases. 1160 8

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.
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PMID:Conformational changes that occur during M3 muscarinic acetylcholine receptor activation probed by the use of an in situ disulfide cross-linking strategy. 1169 1

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond in forming this GSH adduct. The lack of isotope effect in forming the aldoxime from [13CD2]DPC 602 suggests its formation does not occur through the imine intermediate. Instead the data supports the postulated mechanism of hydroxylamine and nitroso intermediates as precursors to the aldoxime. However, the formation of the GSH adduct from [13CD2]DPC 602 did show a significant intramolecular kinetic isotope effect (kH/kD = 2.3) since a carbon-deuterium bond had to be broken on the aldoxime prior to the formation of the adduct. A stable nitrile oxide derived from DPC 602 was postulated as the reactive intermediate responsible for forming this unique GSH adduct.
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PMID:P450-mediated metabolism of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)- [1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole- 5-carboxamide (DPC 423) and its analogues to aldoximes. Characterization of glutathione conjugates of postulated intermediates derived from aldoximes. 1180 May 98

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).
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PMID:Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor. 1205 96

A novel prothrombin activator enzyme, which we have named 'berythractivase', was isolated from Bothrops erythromelas (jararaca-da-seca) snake venom. Berythractivase was purified by a single cation-exchange-chromatography step on a Resource S (Amersham Biosciences) column. The overall purification (31-fold) indicates that berythractivase comprises about 5% of the crude venom. It is a single-chain protein with a molecular mass of 78 kDa. SDS/PAGE of prothrombin after activation by berythractivase showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin, independent of the prothrombinase complex. Chelating agents, such as EDTA and o -phenanthroline, rapidly inhibited the enzymic activity of berythractivase, like a typical metalloproteinase. Human fibrinogen A alpha-chain was slowly digested only after longer incubation with berythractivase, and no effect on the beta- or gamma-chains was observed. Berythractivase was also capable of triggering endothelial proinflammatory and procoagulant cell responses. von Willebrand factor was released, and the surface expression of both intracellular adhesion molecule-1 and E-selectin was up-regulated by berythractivase in cultured human umbilical-vein endothelial cells. The complete berythractivase cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 2330 bp and encodes a preproprotein with significant sequence similarity to many other mature metalloproteinases reported from snake venoms. Berythractivase contains metalloproteinase, desintegrin-like and cysteine-rich domains. However, berythractivase did not elicit any haemorrhagic response. These results show that, although the primary structure of berythractivase is related to that of snake-venom haemorrhagic metalloproteinases and functionally similar to group A prothrombin activators, it is a prothrombin activator devoid of haemorrhagic activity. This is a feature not observed for most of the snake venom metalloproteinases, including the group A prothrombin activators.
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PMID:A prothrombin activator from Bothrops erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning. 1222 92

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.
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PMID:Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor. 1259 66

Antithrombin and its cofactor, heparin, target both the product of prothrombin activation by prothrombinase, thrombin, as well as the enzyme responsible for the reaction, factor (F)Xa. These studies were carried out to quantify the effects of each of the prothrombinase components on the half-life of FXa in the presence of antithrombin and the low-molecular-weight heparins (enoxaparin, Aventis, Laval, Quebec, Canada) or the heparin pentasaccharide (fondaparinux, Organon Sanofi-Synthelabo, Cypress, TX, USA). Experiments were carried out using a recombinant form of prothrombin in which the active site serine has been mutated to cysteine and subsequently labeled with fluorescein. This mutant allowed calculation of the second order rate constant for inhibition of FXa by antithrombin in such a way that competition for antithrombin by thrombin is eliminated and competition for FXa by prothrombin is accounted for. Intrinsic rate constants for the inhibition of FXa by antithrombin-enoxaparin and antithrombin-fondaparinux, in the presence of the various prothrombinase components, were calculated. Addition of phospholipid had no significant effect on the second order rate constant for inhibition of FXa by antithrombin, while addition of FVa appeared to be mildly protective. Further addition of prothrombin however, caused profound protection of FXa, increasing its half-life from 1.1 to 353 s in the case of fondaparinux, and from 0.4 to 42 s in the case of enoxaparin. Similar results were reported for unfractionated heparin previously [1]. Therefore, in the presence of unfractionated heparin, fondaparinux, or enoxaparin, prothrombinase is profoundly protected from antithrombin.
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PMID:Factor Xa is highly protected from antithrombin-fondaparinux and antithrombin-enoxaparin when incorporated into the prothrombinase complex. 1287 28

A structure has been proposed for glucose transporter-1 (GLUT1) based upon homology modeling that is consistent with the results of numerous mutagenesis studies (Mueckler, M., and Makepeace, C. (2004) J. Biol. Chem. 279, 10494-10499). To further test and refine this model, the relative orientation and proximity of transmembrane helices 4 and 8 were analyzed by chemical crosslinking of di-cysteine mutants created in a reporter GLUT1 construct. All six native cysteine residues of GLUT1 were changed to either glycine or serine residues by site-directed mutagenesis, resulting in a functional Glut1 construct with Cys mutated to Gly/Ser (C-less). The GLUT1 reporter molecule was engineered from C-less GLUT1 by creating a unique cleavage site for factor Xa protease within the central cytoplasmic loop and by eliminating the site of N-linked glycosylation. Fourteen functional di-cysteine mutants were then created from the C-less reporter construct, each mutant containing a single cysteine residue in helix 4 and one cysteine residue in helix 8. These mutants were expressed in Xenopus oocytes, and the sensitivity of each mutant to intramolecular crosslinking by two homo-bifunctional, thiol-specific crosslinking reagents, bismaleimidehexane and 1,4-phenylenedimaleimide, was ascertained by protease cleavage followed by immunoblot analysis. Four pairs of cysteine residues, Cys(148)/Cys(328), Cys(145)/Cys(328), Cys(148)/Cys(325), and Cys(145)/Cys(325), were observed to be in close enough proximity to be susceptible to crosslinking by one or both reagents. All five of the cysteine residues susceptible to crosslinking are predicted to lie on the same face of helix 4 or 8 and to reside close to the cytoplasmic face of the membrane. These data indicate that the cytoplasmic ends of helices 4 and 8 lie within 6-16 A of one another and that the two helices twist or tilt such that they are further than 16 A apart toward the center and the exoplasmic side of the membrane. An updated model for the clustering of the transmembrane helices of GLUT1 is presented based on these data.
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PMID:Relative proximity and orientation of helices 4 and 8 of the GLUT1 glucose transporter. 1507 87

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.
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PMID:Expression, refolding, and purification of recombinant human granzyme B. 1559 56


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