Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.37 (neutrophil elastase)
 4,078 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.
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PMID:Endocytosis and degradation of alpha 1-antitrypsin-protease complexes is mediated by the serpin-enzyme complex (SEC) receptor. 221 87

Peptides containing alpha-aminoboronic acids with neutral side chains are highly effective reaction intermediate analog inhibitors of the serine proteases leukocyte elastase, pancreatic elastase, and chymotrypsin. A protocol has been developed for the synthesis of peptides containing alpha-aminoboronic acids with a basic, 3-guanidinopropyl side chain (boroArg) to extend the range of these compounds to trypsin-like proteases. Ac-(D)Phe-Pro-boroArg-OH, Boc-(D)Phe-Pro-boroArg-OH, and H-(D)Phe-Pro-boroArg-OH were prepared as inhibitors of thrombin based on earlier observations that it has a high affinity for this sequence. All three boronic acids are highly effective, slow-binding inhibitors of thrombin, inhibiting it with final inhibition constants and association rates of: 41 pM, 5.5 x 10(6) M-1 s-1; 3.6 pM, 9.3 x 10(6) M-1 s-1; less than 1 pM, 8.0 x 10(6) M-1 s-1, respectively. Comparison of their binding at equilibrium to thrombin, plasma kallikrein, factor Xa, plasmin, and two-chain tissue plasminogen activator has shown that all three inhibitors have at least 2 orders of magnitude greater affinity for thrombin, with the exception of the acetyl derivative which has a 40-fold greater affinity for thrombin than kallikrein. The boroarginine peptides are effective in inhibiting the action of thrombin in rabbit plasma against its physiological substrates. Activated partial thromboplastin time was significantly prolonged in vitro by all of the inhibitors at concentrations of 50-200 nM. Prolongations of activated partial thromboplastin time were also observed in rabbits after intravenous (40-80 micrograms/kg or subcutaneous (0.20-2 mg/kg) injections of Ac-(D)Phe-Pro-boroArg-OH. Results indicate that this new class of synthetic thrombin inhibitors may be clinically useful as antithrombotic agents.
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PMID:The selective inhibition of thrombin by peptides of boroarginine. 221 2

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

Granulocyte elastase (GE, EC 3.4.21.37) is a key enzyme in tissue injury. To elucidate the role of GE in tissue injury, a new method of measuring GE activity in various inflammatory tissue fluids was developed using diazotization and the chromogenic synthetic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (S-2482). GE activity demonstrated first order kinetics in the range from 1.9 to 30 U/l. Other proteases, such as pancreatic elastase, trypsin, and chymotrypsin did not hydrolyze S-2484. This assay permits the determination of GE activity with a coefficient of variance less than 7.8% and 95.6 to 105.4% recovery. With this method, hydrolytic GE activity was found to be increased in bronchoalveolar lavage fluid from patients with ARDS or pneumonia, synovial fluid from patients with rheumatoid arthritis, and blister fluid from burn patients.
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PMID:A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. 231 34

A potent inhibitor of human leukocyte elastase (EC 3.4.21.37) and porcine pancreatic elastase (EC 3.4.21.36) was purified to homogeneity from human horny layers. It inhibits human leukocyte elastase and porcine pancreatic elastase in a 1:1 molar ratio and shows equilibrium dissociation constants of 6 x 10(-10) M and 1 x 10(-9) M, respectively. Inhibition of plasmin, trypsin, alpha-chymotrypsin, and cathepsin G was not observed. This inhibitor proved to be an acid stable basic peptide with an isoelectric point of 9.7. The complete amino acid sequence appears to be unique with 38% homology to the C-terminal half of antileukoprotease. The sequence shows that the inhibitor is composed of 57 amino acids and predicts a Mr of 7017. The high affinity as well as the apparent specificity for elastases suggests a functional role in preventing elastase-mediated tissue proteolysis. It is suggested that the term "elafin" be used to designate this inhibitor.
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PMID:Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. 239 96

Monitoring of plasma proteinases, proteinase inhibitors and other selective plasma proteins was evaluated in patients undergoing Y-graft aortofemoral bypass operation. Fast-reacting acute-phase proteins (C-reactive protein, antichymotrypsin, alpha 1-acid glycoprotein) and slow-reacting proteins (haptoglobin, alpha 1-antitrypsin) increased significantly 48-120 h after operation. By contrast, no significant increase was found between plasma ceruloplasmin levels before clamping and after declamping. Activity and concentration of alpha 2-macroglobulin decreased postoperatively and remained significantly lowered throughout the observation period. Plasma levels of granulocyte elastase were elevated significantly 1 h after declamping, whereas trypsin-binding capacity decreased immediately after the release of the clamp. Aprotinin pretreatment caused higher trypsin-binding capacity of the plasma, significantly lower 'unspecific' proteolytic (azocasein-hydrolyzing) activity and significantly lower non-TCA precipitable low molecular weight plasma protein concentration. Our results confirm the data of several authors that monitoring of plasma proteinases, proteinase inhibitors and other selective plasma proteins may be helpful in evaluating surgical patients postoperatively.
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PMID:Plasma proteinases, proteinase inhibitors and other selective plasma proteins following aortofemoral bypass operation. 242 35

A synthetic gene encoding the protein sequence of mature bovine pancreatic trypsin inhibitor (BPTI) has been cloned into a novel E. coli expression vector. After in vitro gene amplification by successive DNA duplications, more than 600 000 mostly inactive inhibitor molecules may be recovered from a single cell. After purification the inhibitory activity can be reconstituted almost completely. The specificity of BPTI for trypsin is abolished by a single amino acid exchange from lysine to isoleucine at position 15. The altered protein is shown to be an efficient inhibitor of human leukocyte elastase.
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PMID:A synthetic operon containing 14 bovine pancreatic trypsin inhibitor genes is expressed in E. coli. 243 25

Human alpha 2-antiplasmin rapidly forms a stable, equimolar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reactive site of the inhibitor with the concomitant release of a small peptide fragment which apparently represents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of alpha 2-antiplasmin encompasses a P1-P'1 Arg-Met sequence. However, unlike alpha 1-1-proteinase inhibitor which has a Met residue in the P1-position, oxidation of alpha 2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mechanistic importance of the P'1-residue during enzyme inactivation by this inhibitor.
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PMID:The reactive site of human alpha 2-antiplasmin. 243 12

Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.
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PMID:Mechanism of action of inter-alpha-trypsin inhibitor. 244 Apr 71

The semisynthesis of homologues of aprotinin (BPTI) is described. The P1 amino acid residue of these homologues was substituted by other amino acids using peptide synthetic methods. The reactive-site-modified inhibitor (with the Lys15-Ala16 peptide bond hydrolyzed) was used as starting material. All carboxyl groups of the modified inhibitor were esterified with methanol, then the Lys15 methyl ester group was hydrolyzed selectively. Afterwards, Lys15 itself was split off. A new amino acid residue was incorporated by using water-soluble carbodiimide combined with an acylation catalyst. tert-Butyl-ester-protected amino acids were used for reinsertion. The method was tested by re-insertion of Lys15 to reconstitute the original inhibitor. Thirteen BPTI homologues with coded (Lys, Glu, Gly, Ala, Val, Ile, Leu) or uncoded amino acids (Abu, Ape, aIle, Ahx, tLeu, Neo) in position 15 were synthesized and the specificity of the inhibitors investigated. Amongst these, [Val15]BPTI was shown to be an excellent inhibitor for human polymorphonuclear leukocyte elastase having a complex dissociation constant of 0.11 nM. This inhibitor showed no detectable affinity to bovine pancreatic trypsin.
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PMID:Preparation of chemically 'mutated' aprotinin homologues by semisynthesis. P1 substitutions change inhibitory specificity. 245 25


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