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:3.4.21.37 (neutrophil elastase)
4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver cells (the C-9 cell line) are stimulated to metabolize arachidonic acid by alpha-thrombin, its receptor polypeptide, gamma-thrombin, and trypsin. Prostaglandin (PG) I2 synthesis stimulated by alpha-thrombin is inhibited by dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA), by hirudin, by the synthetic tyrosine-sulfated dodecapeptide corresponding to residues 53-64 of hirudin (hirugen), by the Tyr(SO3H)63-hirudin fragment 54-65 and by rabbit lung thrombomodulin. Stimulation of arachidonic acid metabolism by the receptor octapeptide, SFLLRNPN, is not affected by DAPA or hirudin. gamma-Thrombin stimulates arachidonic acid metabolism but at 300 to 400-fold higher concentrations. Trypsin stimulates arachidonic acid metabolism. Trypsin's proteolytic activity is required--its ability to stimulate is abolished if it is incubated with Na-p-tosyl-L-lysine chloromethyl ketone (TLCK) or bovine pancreatic trypsin inhibitor. Prior treatment of the rat liver cells with alpha-thrombin blocks subsequent stimulation by alpha-thrombin, but not by trypsin, whereas prior treatment with trypsin blocks subsequent stimulation by trypsin, but not the activity stimulated by alpha-thrombin. Prior treatment of the cells with the serine-proteases, chymotrypsin, pancreatic or neutrophil elastase and thrombocytin from Bothrups atrox venom, block alpha-thrombin's activation of PGI2 production, but not the activity stimulated by trypsin. These findings indicate that alpha-thrombin and trypsin stimulate PGI2 production via different receptors.
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PMID:Alpha-thrombin and trypsin use different receptors to stimulate arachidonic acid metabolism. 793 15

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99

alpha 1-Antitrypsin is a circulating serine proteinase inhibitor that protects the lungs against proteolysis by the enzyme neutrophil elastase. Most northern Europeans have only the normal M form, but some 4% are heterozygotes for the Z deficiency mutant. This mutant is characterized by the substitution of a positively charged lysine residue for a negatively charged glutamic acid at position 342 and results in normal gene translation but reduced protein secretion into the plasma. The plasma levels of antitrypsin in homozygotes are only 15% of normal, the other 85% being retained in the endoplasmic reticulum of the hepatocyte. This review describes the effect of the Z mutation on the structure and function of antitrypsin and illustrates the importance of understanding protein structure in solving the mechanism of Z antitrypsin retention within the liver. We demonstrate that antitrypsin accumulation in the liver results from a unique interaction between antitrypsin molecules. The Z mutation perturbs the gap between the third and fifth strands of the A sheet, allowing the reactive center loop of one molecule to insert into the A sheet of a second. This loop-sheet polymerization results in the formation of chains of protein which form insoluble inclusions in the endoplasmic reticulum, resulting in hepatocellular damage and cirrhosis. In addition, the Z mutation results in a distortion of the circular dichroic spectrum, a rearrangement of the reactive center loop with respect to the A sheet, and a reduction in association rate constant with the cognate proteinase neutrophil elastase.
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PMID:A protein structural approach to the solution of biological problems: alpha 1-antitrypsin as a recent example. 821 81

Nitrogen dioxide (NO2), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. In order to gain information on the mechanism by which NO2 damages the lung and proteins vital to its function, as well as its reaction with proteins in general, in vitro exposures of alpha-1-proteinase inhibitor (alpha 1PI), elastin, poly-L-lysine, and poly-L-arginine were performed. The ability of alpha 1PI to inhibit its natural physiological target, human neutrophil elastase (HNE), declined with exposure to 54% of the control value at molar ratios of NO2:alpha 1PI of 400:1 and greater. Exposure of alpha 1PI to NO2 resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO2:alpha 1PI of 100:1 and greater. The results of parallel O-phthalaldehyde and bicinchoninic acid protein assays as well as amino acid analysis on control and NO2-exposed alpha 1PI suggested a reactivity of NO2 with lysine residues. Elastin and poly-L-lysine were labeled by reductive methylation of amino groups with [3H]HCHO prior to treatment with NO2 in aqueous solutions at physiological pH. NO2 exposure of elastin resulted in the solubilization of 84% of the associated radioactivity of which 79% was identified as [3H]methyllysine by amino acid analysis. After NO2 exposure of poly-L-[3H]lysine, gel filtration chromatography revealed that the 50,000 M(r) poly-L-[3H]lysine had been degraded to small peptides of 1-3000 M(r). Similarly, after NO2 exposure of unlabeled poly-L-arginine, gel filtration chromatography, and total peptide analysis revealed that the 47,500 M(r) peptide was also partially degraded to peptides. These results suggest that NO2 reacts with the epsilon-amino groups of Lys residues (primary amines) and with the amide nitrogen (secondary amines) of surface-exposed Lys and Arg residues in the peptide backbone to result in peptide bond cleavage. These findings are the first indication of NO2-mediated peptide degradation and provide additional data on the potential of NO2 to damage proteins vital to the function of the lung in an in vitro exposure system.
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PMID:Nitrogen dioxide reactivity with proteins: effects on activity and immunoreactivity with alpha-1-proteinase inhibitor and implications for NO2-mediated peptide degradation. 832 82

Fibrin thrombi form at sites of injury, where leukocytes release a variety of oxidants. To determine whether oxidants might affect proteins of the fibrinolytic system, we examined the effects of various oxidants on plasmin. Plasmin was not inhibited by micromolar concentrations of hypochlorous acid, chloramine T, or H2O2. Neither Fe nor Cu affected plasmin alone or in the presence of H2O2. However, incubation of plasmin with 5 mumol/L Cu(I or II) in the presence of the reducing agent ascorbic acid resulted in a loss of its hydrolytic activity towards proteins as well as towards small synthetic substrates. The addition of EDTA, but not mannitol, prevented its inactivation. Inactivation was prevented by the addition of catalase and accelerated by hydrogen peroxide. Preincubation of plasmin with the competitive inhibitor alpha-N-acetyl-L-lysine methyl ester prevented inactivation by Cu(II) and ascorbate. These results together suggest site-specific oxidation of plasmin's active site. Treatment of the plasminogen activators tissue plasminogen activator and two-chain urokinase-type plasminogen activator, as well as trypsin, neutrophil elastase, and thrombin with Cu(II) and ascorbate resulted in a loss of their amidolytic and proteolytic activity, indicating the general susceptibility of serine proteases to this type of oxidation. Oxidation of the zymogens Glu-plasminogen and single-chain urokinase-type plasminogen activator by Cu(II) and ascorbate resulted in the failure of these molecules to generate active enzymes when treated with plasminogen activators or plasmin, respectively. The active site His residue may be the target of oxidative inactivation, as evidenced by the partial protection afforded plasmin by the addition of Zn(II), histidine, or the platinum derivative, platinum(II) (2,2':6',2"-terpyridine) chloride. Because platelets contain micromolar concentrations of Cu and leukocytes are rich in ascorbate, Cu-dependent site-specific oxidation might play a role in modulating proteolytic events and the life span of thrombi formed at sites of tissue injury.
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PMID:Oxidative inactivation of plasmin and other serine proteases by copper and ascorbate. 836 3

ICI 200,880, 4-(4-chlorophenylsulphonylcarbamoyl)benzoyl-L-valyl-L-proline-1(RS )-(1-trifluoroacetyl-2-methylpropyl)amide (I), is a human neutrophil elastase inhibitor in development by ICI Pharmaceuticals Group. A specific and sensitive radioimmunoassay has been developed for this compound in human serum. An hydroxysuccinimide ester analogue of ICI 200,880 was coupled to the lysine residues of bovine serum albumin, characterized by gel electrophoresis and injected into rabbits to stimulate antibody formation; a highly specific, high titre antibody was obtained. An 125I-diiodinated 4-hydroxy-phenyl derivative of ICI 200,880 was utilized as the radiolabelled antigen. Satisfactory zero and non-specific binding were achieved with a 2-h pre-incubation of ICI 200,880 and antiserum at 37 degrees C, followed by the addition of radiolabelled antigen and a second 2-h incubation. Separation of bound and free radiolabel was achieved by employing PEG-goat anti-rabbit IgG separant. Sensitivity of 25 pg ml-1 ICI 200,880 in human serum was achieved (n = 12), with quantitative recovery of 106%, inter-assay precision of 6.3% and an average relative binding statistically different than that of pooled human serum. Serum quality control samples spiked at 600 and 100 pg ml-1 ICI 200,880 averaged 104% recovery over 6 validation days, with intra-assay precision of 12.8% RSD and inter-assay precision of 9.2% RSD (n = 24). Cross-reactivity of the ICI 200,880 antibody to three known metabolites and several analogues was negligible. Over 1200 clinical samples following aerosol administration of ICI 200,880 have been analysed by this procedure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A specific radioimmunoassay for the measurement of ICI 200,880, an elastase inhibitor, in human serum. 839 28

A major feature of the structure of alpha 1-antitrypsin is a five-stranded A-sheet into which the reactive center loop inserts after cleavage. We describe here the effect of the Z mutation (342Glu to Lys) at the head of the fifth strand of the A-sheet on the mobility of the reactive center loop and hence on the physical properties of the antitrypsin molecule. The mutant Z but not the normal M antitrypsin spontaneously polymerizes at 37 degrees C by a mechanism involving the insertion of the reactive center loop of one molecule into the A-sheet of a second. It is demonstrated that Z antitrypsin polymerized after incubation with 1.0 M guanidinium chloride at 37 degrees C at the same rate as M antitrypsin. Reducing the temperature to 4 degrees C favored the formation of the L-state in M antitrypsin in which the loop is stably incorporated into the A-sheet, but resulted in loop-sheet polymerization in Z antitrypsin. Z, like M antitrypsin, undergoes the S to R transition, but we show that the accompanying change in thermal stability results from loop-sheet polymerization (S) which can be prevented by the insertion of the cleaved strand of the reactive center loop into the A-sheet (R). Z antitrypsin has a reduced association rate constant with neutrophil elastase [(5.3 +/- 0.06) x 10(7) and (1.2 +/- 0.02) x 10(7) M-1 s-1 for M and Z, respectively], but both M and Z antitrypsin had Ki values of less than 5 pM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of the Z mutation on the physical and inhibitory properties of alpha 1-antitrypsin. 842 59

In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinases: bovine alpha chymotrypsin A, porcine pancreatic elastase, subtilisin Carlsberg, Streptomyces griseus proteinases A and B, and human leukocyte elastase. Eight of the Xs are coded amino acids: Ala, Ser, Val, Met, Gln, Glu, Lys, and Phe, and five are noncoded: Abu, Ape, Ahx, Ahp, and Hse. They were chosen to simplify the interamino acid comparisons. In the homologous series of straight-chain side chains Ala, Abu, Ape, Ahx, Ahp, free energy of binding decreases monotonically with the side-chain length for chymotrypsin with large binding pocket, but even for this enzyme shows curvature. For the two S. griseus enzymes a minimum appears to be reached at Ahp. A minimum is clearly evident for the two elastases, where increasing the side-chain length from Ahx to Ahp greatly weakens binding, but much more so for the apparently more rigid pancreatic enzyme than for the more flexible leukocyte enzyme. beta-Branching (Ape/Val) is very deleterious for five of the six enzymes; it is only slightly deleterious for the more flexible human leukocyte elastase. The effect of gamma-branching (Ahx/Leu), of introduction of heteroatoms (Abu/Ser), (Ape/Hse), and (Ahx/Met), and of introduction of charge (Gln/Glu) and (Ahp/Lys) are tabulated and discussed. An important component of the free energy of interaction is the distortion of the binding pocket by bulky or branched side chains. Most of the variants studied were obtained by enzymatic semisynthesis. X18 variants of the 6-18 peptide GlyNH2 were synthesized and combined with natural reduced peptide 19-56. Disulfide bridges were formed. The GlyNH2 was removed and the reactive-site peptide bond X18-Glu19 was synthesized by complex formation with proteinase K. The resultant complexes were dissociated by sudden pH drop. This kinetically controlled dissociation afforded virgin, reactive-site-intact inhibitor variants.
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PMID:Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues. 849 99

In a previous report, we described the molecular cloning, expression, and partial characterization of a second human tissue factor pathway inhibitor (TFPI), which we designated as TFPI-2 [Sprecher, C. A., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3353-3357]. Recombinant TFPI-2 inhibited the amidolytic activity of trypsin as well as that of factor VIIa in complex with tissue factor. TFPI-2 recently has been shown to be identical to placental protein 5 (PP5), a glycoprotein originally isolated from placenta that exhibits serine protease inhibitory activity. In the present study, we have examined TFPI-2/PP5 for its ability to inhibit a number of serine proteases involved in blood coagulation and fibrinolysis, inasmuch as TFPI-2/PP5 prolonged the coagulation time of human plasma induced by either tissue factor or contact activation in a dose-dependent manner. In addition to its ability to inhibit the amidolytic and proteolytic activities of the factor VIIa-tissue factor complex, TFPI-2/PP5 strongly inhibited the amidolytic activities of human factor XIa, human plasma kallikrein, and human plasmin with Ki values of 15, 25, and 3 nM, respectively. TFPI-2/PP5 was also a weak inhibitor of the activation of factor X by a complex of human factor IXa and poly(lysine) with an apparent Ki of 410 nM. Heparin markedly enhanced the ability of TFPI-2/PP5 to inhibit factor VIIa-tissue factor both in the solution phase and on cell surfaces. In addition, heparin augmented the inhibition of human factor Xa amidolytic activity at relatively high levels (10-100 nM) of TFPI-2/PP5. No significant inhibition of glandular kallikrein, urinary plasminogen activator, tissue plasminogen activator, human activated protein C, human factor Xa, human thrombin, or leukocyte elastase was observed when these proteases were incubated with TFPI-2 in the absence of heparin.
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PMID:Inhibitory properties of a novel human Kunitz-type protease inhibitor homologous to tissue factor pathway inhibitor. 855 84

We have previously shown that a functional free apolipoprotein[a] (apo[a]) can be isolated from its parent lipoprotein[a] (Lp[a]) by a mild reductive procedure. To shed further light on the properties of Lp[a] and apo[a] we subjected them to a limited proteolysis by porcine pancreatic elastase. This enzyme cleaved both at the Ile3520-Leu3521 bond in the linker between kringles IV-4 and IV-5 of apo[a] generating two fragments F1 and F2. In contrast to F1, which represented the N-terminal portion of apo[a] and was functionally inert, F2, representing the C-terminal domain, bound to lysine-Sepharose, fibrinogen, and fibronectin and formed a miniLp[a] particle when incubated with LDL. The proteolytic pattern by pancreatic elastase was also exhibited by human leukocyte elastase. F1, injected intravenously into normal mice, was rapidly cleared (Ty2, 2.9 h) and after 1 h fragments in the size range of 100-33 kDa were observed in the urine. In turn, F2 had a longer residence time (Ty2, 5 h) and was excreted in the urine only after 5 h as fragments of 70-45 kDa. Fragments in the same size range as found after F1 injection were also present in the urine after injection of apo[a] or Lp[a]. Moreover, apo[a] fragments of the size seen in mouse urine were spontaneously present in normal human urine and appeared derived from larger apo[a] fragments in the plasma. Our results indicate that enzymes of the elastase family cleave human apo[a] in vitro into two main fragments that differ in structural and functional properties and metabolic behavior. The comparable size of apo[a] fragments observed in the urine of humans and injected mice invites the speculation that enzymes of the elastase family may play a role in the biology of Lp[a] in vivo.
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PMID:Functional and metabolic differences between elastase-generated fragments of human lipoprotein[a] and apolipoprotein[a]. 886 63


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