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)

Low molecular mass heparin (5.1 kDa) forms a tight complex with mucus proteinase inhibitor, the physiologic neutrophil elastase inhibitor of the upper respiratory tract. This binding strongly enhances the intrinsic fluorescence of the inhibitor and the rate of neutrophil elastase inhibitor association. One mole of this heparin fragment binds 1 mol of inhibitor with a Kd of 50 nM. From the variation of Kd with ionic strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy i due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. strength, it is inferred that (i) 85% of the heparin--inhibitor binding energy is due to electrostatic interactions, (ii) about seven ionic interactions are involved in heparin--inhibitor binding. and (iii), about one-third of low quantum yield of Trp30, the single tryptophan residue of the inhibitor, blue-shifts its maximum emission wavelength by 6 nm, decreases the acrylamide quenching rate constant by a factor of 4, and increases the mean intensity weighted lifetime by a factor of 2.5. These important spectroscopic changes evidence a heparin--induced conformational change of the inhibitor which buries Trp30 in a very hydrophobic environment. Heparin accelerates the inhibition of elastase in a concentration-dependent manner. When both enzyme and inhibitor are saturated by the polymer, the second-order association rate constant is 7.7 x 10(7) M-1 s-1, a value that is 27-fold higher than that measured with the free partners. This finding may have important physiologic and therapeutic bearing.
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PMID:Heparin-induced conformational change and activation of mucus proteinase inhibitor. 152 65

Exposure of purified human plasma fibronectin to the myeloperoxidase-H2O2-Cl- system of neutrophils or to reagent HOCl resulted in extensive changes to its primary and tertiary structures. When 1.14 microM fibronectin was exposed to 50-400 microM HOCl or 50-400 microM H2O2 plus myeloperoxidase and Cl-, there was progressive loss of tryptophan fluorescence and cysteines, and an increase in bityrosine fluorescence and carbonyl content. Analysis by SDS-PAGE indicated extensive crosslinking of the fibronectin, the crosslinks being stable under reducing conditions. The coincident increase of bityrosine fluorescence suggests that crosslinking may be largely due to intermolecular bityrosines rather than disulfides. All changes observed with the myeloperoxidase system were inhibited by azide or methionine, and were dependent upon the presence of chloride, indicating that they are mediated by HOCl. The reaction between HOCl and fibronectin resulted in the formation of long-lived chloramines. Exposure to increasing amounts of oxidant resulted in an increase in the susceptibility of fibronectin to proteolytic attack by purified neutrophil elastase. Analysis by SDS-PAGE showed a different fragmentation pattern for oxidant-treated fibronectin compared with the native protein. This suggests that regions of the molecule which were previously resistant to proteolysis were denatured to create susceptible sites for elastase. This demonstration that fibronectin is extensively modified by the myeloperoxidase system has implications for the mechanism of tissue injury by neutrophils in inflammation, since a loss of functional fibronectin would result in cell detachment and a distortion of normal tissue organization.
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PMID:Oxidative damage to fibronectin. I. The effects of the neutrophil myeloperoxidase system and HOCl. 184 32

Human neutrophil elastase has an extended hydrophobic substrate binding site which serves as a target for a number of hydrophobic inhibitors. We show here that the parinaric acids, fluorescent-conjugated tetraenoic fatty acids of plant origin, are inhibitors of neutrophil elastase. cis-Parinaric acid (cis-PA) interacts with the enzyme in two inhibitory modes. The high affinity interaction (Ki = 55 +/- 6 nM) results in partial noncompetitive inhibition of amidolytic activity, with 82% residual activity. A lower affinity interaction with cis-PA (Ki = 4 +/- 1 microM) results in competitive inhibition. trans-PA also acts as a high affinity partial noncompetitive inhibitor of elastase with a Ki equal to that for cis-PA but has no low affinity competitive inhibitory action. The endogenous fluorescence from the 3 tryptophan residues in elastase is partially quenched on binding cis- or trans-PA. Dependence of quenching of tryptophan fluorescence on PA concentration is consistent with binding to a single site with an apparent Kd of 26 +/- 3 nM, which may be equivalent to the high affinity partial noncompetitive inhibitory binding mode. Analysis of quenching according to the modified Forster theory of energy transfer developed by Snyder and Freire (Snyder, B., and Freire, E. (1982) Biophys. J. 40, 137-148) leads to an estimate of apparent closest indole-PA distance of 13 +/- 3 A. Fluorescence of either cis- or trans-PA is apparently unperturbed upon binding in the high affinity mode to elastase, but at micromolar cis-PA concentrations, binding to elastase results in a blue shift and 20% increase in intensity of PA emission, suggesting that the lower affinity competitive inhibitory binding mode of binding to elastase provides a hydrophobic environment for cis-PA.
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PMID:Parinaric acids as probes of binding domains in neutrophil elastase. 186 48

We have modified the single cysteine residue of alpha 1-protease inhibitor (alpha 1-PI) with HgCl2, methylmethane thiosulfonate, oxidized glutathione (GSSG), and N-(1-anilinonaphthyl-4)maleimide (ANM). Whereas native alpha 1-PI combines rapidly and quasi-irreversibly with neutrophil elastase, the thiol-modified alpha 1-PI derivatives are dissociable reversible competitive inhibitors of the enzyme, with values of Ki in the range of 6-7 nM. Removal of the thiol modifications restores the rapid irreversible mode of inhibition. Once native alpha 1-PI has combined with neutrophil elastase, the enzyme-inhibitor complex retains a reactive thiol group, but the two proteins can no longer be dissociated by subsequent reaction with ANM, even after exposure to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. From kinetic measurements of fluorescence, ANM-modified alpha 1-PI combines with neutrophil elastase via an apparent biomolecular process with a second order rate constant on the order of 10(5) M-1 S-1. We estimate a dissociation rate constant on the order of 10(-3) S-1. The emission of ANM-modified alpha 1-PI is increased in intensity and blue shifted from the maximum in ANM-modified cysteine, consistent with a predominantly nonpolar environment. Association with neutrophil elastase results in an additional blue shift with further increase in intensity, consistent with a further decrease in polarity of the environment of the cysteine. Modification with methylmethane thiosulfonate or GSSG results in a small decrease in quantum yield and a red shift in the tryptophan emission spectrum of the modified inhibitor, suggestive of increased polarity of the environment of at least 1 of the 2 tryptophan residues in alpha 1-PI. These changes are reversed by dithiothreitol and are consistent with a conformational change which transforms the inhibitory activity from a rapid, irreversible mode in native alpha 1-PI to a dissociable competitive mode in the mixed disulfide derivatives.
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PMID:Reversible inhibition of neutrophil elastase by thiol-modified alpha-1 protease inhibitor. 200 60

Chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the observed rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.
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PMID:Influence of tryptophan modification upon digestion of antithrombin III by elastase. 205 15

Human neutrophil elastase (HNE) can be inhibited by unsaturated fatty acids, including oleic acid [Ashe, B. M., & Zimmerman, M. (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook, L., & Ternai, B. (1988) Biol. Chem. Hoppe-Seyler 369, 627-631], but is not affected by saturated fatty acids. We have shown that the interaction of oleic acid with HNE can be characterized by two apparent inhibitory modes: a high-affinity mode (Ki = 48 +/- 3 nM), resulting in partial noncompetitive inhibition (87% residual activity), and a competitive inhibitory mode of lower affinity (Ki = 16 +/- 1 microM). Binding of oleate in the high-affinity mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HNE. This shift is maximal in the presence of 1 microM oleate; higher concentrations of fatty acid have no further effect on the fluorescence spectrum. The negatively charged fluorescent ester of oleic acid and hydroxypyrenetrisulfonate (HPTSoleate) interacts with HNE at an apparent single site (Ki = 44 +/- 3 nM), resulting in competitive inhibition. A blue shift in the emission maximum of the pyrene fluorescence at 410 nm and a decrease in the ratio of the intensities of the maximum at 388 and 410 nm indicate that upon binding to HNE the environment of the pyrene ring in HPTSoleate becomes more hydrophobic.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibitors directed to binding domains in neutrophil elastase. 227 33

Based on available knowledge, this study shows that alpha-1-proteinase inhibitor (alpha 1-PI) plays an important role in protecting lung elastin from elastolytic proteinases, particularly human neutrophil elastase (HNE). Studies previous to this one showed that alpha 1-PI was very susceptible to inactivation by oxidants. We sought to use this oxidant sensitivity as an in vivo marker for ozone (O3) and nitrogen dioxide (NO2) exposure. The mechanism of alpha 1-PI inactivation by O3 and NO2 was examined to provide insight concerning the pathogenesis of oxidant-mediated lung damage. Attention also was focused on the bronchial leukocyte proteinase inhibitor (BLPI), which inhibits HNE in the bronchial secretions. Careful examination of blood plasma samples from individuals exposed to 0.5 ppm O3 for four hours on two consecutive days failed to detect any inactivation of alpha 1-PI. This result showed that blood alpha 1-PI was not a satisfactory marker for O3 exposure, but, more importantly, demonstrated that inhaling O3 for short periods does not grossly inactivate this important protein. Studies on BLPI showed that it is a significant inhibitor of HNE and probably plays a more important role in protecting the lung than previously thought. BLPI, like alpha 1-PI, was found to be inactivated by oxidants, including O3 and NO2. The mechanism of O3 inactivation of leukocyte proteinase inhibitors was studied using alpha 1-PI, alpha-1-antichymotrypsin (alpha 1-Achy), BLPI, and Eglin C. While all these inhibitors differed in structure, the concentrations of O3 required for inactivation were essentially the same, except for alpha 1-Achy, which only lost half of its inhibitory activity. It would seem from these results that O3 can damage proteins via the oxidation of any of the following: tryptophan (Trp), methionine (Met), tyrosine (Tyr), or histidine (His) residues. Interestingly, Eglin C, which does not have oxidizable amino acids in its inhibitory active site, was inactivated by the same amount of O3 as BLPI, BLPI was easily inactivated by a methionine-specific oxidant, suggesting an important role for methionine in this inhibitor. In vitro exposure of alpha 1-PI and BLPI to 800 moles of NO2 per mole of inhibitor resulted in 35% and 50% losses of HNE inhibitory activity, respectively. Tryptophan was destroyed by NO2 and studies are in progress to examine effects on other amino acids.
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PMID:Effects of ozone and nitrogen dioxide on human lung proteinase inhibitors. 326 87

Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor (alpha 1-PI) both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tyrosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes witrh serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.
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PMID:Ozone inactivation of human alpha 1-proteinase inhibitor. 690 14

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.
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PMID:Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor. 819 13

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.
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PMID:The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C. 1036 30


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