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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

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

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 [Arg15,Glu52]aprotinin gene has been constructed from a synthetic [Glu52]-aprotinin gene via an exchange of the appropriate DNA cassette. The gene has been fused to the N-terminal part of the bacteriophage MS-2 polymerase and expressed in a temperature inducible E. coli expression system. The produced fusion protein is deposited as inclusion bodies. Pure and functionally active [Arg15,Glu52]aprotinin has been obtained after cleavage of the purified fusion protein and renaturation of the aprotinin homologue. Recombinant [Arg15,Glu52]aprotinin shows good inhibition of human anionic and cationic trypsin (Ki less than or equal to 10(-11)M) and of human plasma kallikrein (Ki = 3.2 x 10(-10)M). The inhibition constants for human plasmin are Ki = 1.3 x 10(-10)M and for human urinary kallikrein Ki = 10(-11)M. No inhibition was found with the human proteinases thrombin, coagulation factor Xa, urokinase, tissue plasminogen activator, cathepsin G, leukocyte elastase and pancreatic elastase.
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PMID:Expression, isolation and characterization of recombinant [Arg15,Glu52]aprotinin. 246 33

Aprotinin derivatives with decarboxylated lysine, arginine or valine at position 15, the P1 position of modified aprotinin, were produced semisynthetically. Modified aprotinin with oxidatively deaminated Arg1 and Ala16 was also synthesized. Specific reduction of this derivative yielded a modified aprotinin with lactic acid at position 16, the P'1 position. Only the aprotinin derivatives with decarboxylated Lys15 or Arg15 showed moderate inhibitory activity against trypsin and kallikrein, despite the absence of the carboxyl group. The KD values measured were in the range of 10(-7) M. The aprotinin derivative with decarboxylated valine showed no inhibitory activity; neither against trypsin, kallikrein and chymotrypsin, nor against the human leukocyte elastase. From these data it was concluded that the ion-pair interaction of the Lys15, or the Arg15 inhibitor side-chain with the aspartate in the trypsin specificity pocket is important for the inhibitory activity. Furthermore, the KD values indicated that the interaction of the reactive-site's carbonyl group with the enzyme's oxyanion hole also contributes to the inhibitory activity. These two interactions are important, but not essential for inhibitory activity. In contrast to these findings, the existence of an alpha-amino group at the P'1 position seems to be essential for inhibitory activity. The synthesized aprotinin derivatives lacking an alpha-amino group at this position were without any inhibitory activity against serine proteinases.
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PMID:Semisynthetic aprotinin derivatives with specific alterations at the reactive-site peptide bond can be used to study structure-function relationships. 247 74

Bovine pancreatic trypsin inhibitor (BPTI, also known as aprotinin or Kunitz inhibitor, a mini-protein composed of 58 amino-acid residues, containing a single methionine residue at position 52) has been selectively oxidized by treatment with chloramine T, under mild conditions, to the methionyl sulfoxide derivative. Spleen inhibitor II (SI II, an isoform of BPTI containing two methionine residues at positions 18 and 52) has been oxidized under the same conditions. Oxidation affects the functional properties of the two inhibitors differently: the antiproteolytic activity of BPTI towards bovine trypsin and chymotrypsin, porcine kallikrein and human leukocyte elastase is not changed upon oxidation, while in the oxidized SI II, the affinity for both chymotrypsin and elastase decreases, with respect to the native protein. These results have been directly related to the oxidation of Met18 in SI II, located at the P'3 site in the contact area with the proteases.
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PMID:Selective oxidation of methionine residues in Kunitz-type protease inhibitors. 247 60

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.
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PMID:Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors. 249 9

Nonporous, microparticulate, monodisperse silicas with particle diameters between 0.7 and 2.1 microns are introduced as stationary phases in high-performance affinity chromatography. The immobilization of m-aminophenylboronic acid, p-aminobenzamidine, tri-L-alanine, and concanavalin A onto these silicas was successfully achieved using 3-isothiocyanatopropyl-triethoxysilane as an activation reagent. Immobilized phenylboronic acid was applied to the isolation of nucleosides, nucleotides, and glycoprotein hormones such as bovine follicotropin and human chorionic gonadotropin, while immobilized benzamidine was employed for the isolation of the serine proteases thrombin and trypsin, immobilized tri-L-alanine for the separation of pig pancreatic elastase and human leukocyte elastase, and immobilized concanavalin A for the isolation of horseradish peroxidase. In all affinity chromatographic systems studied, the nonporous monodisperse silicas showed improved chromatographic performance compared to results obtained with porous silica supports using identical activation and immobilization procedures. Furthermore, frontal analysis was used as a method to evaluate the influence of experimental parameters on biological activity and accessible ligand densities. Only minor changes in bioactivity were found with the nonporous affinity supports, where accessibilities were typically higher than ca. 60%. The immobilization of affinity ligands onto porous supports as used in this and associated papers thus represents a successful general procedure for the preparation of stable matrices with fast kinetics for use in high-performance affinity chromatography.
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PMID:High-performance liquid affinity chromatography with phenylboronic acid, benzamidine, tri-L-alanine, and concanavalin A immobilized on 3-isothiocyanatopropyltriethoxysilane-activated nonporous monodisperse silicas. 254 22

Ten dextran sulphates and six chitosan sulphates of variable Mr and extent of sulphate substitution have been examined for their ability to inhibit human leukocyte elastase (HLE). All were potent partial non-competitive inhibitors of this enzyme, highest activity being obtained with compounds of large molecular weight and maximum sulphate incorporation (Ki = 5.0 X 10(-10)M]. In all cases, the dextran sulphates were more effective inhibitors than chitosan sulphates of similar size and charge, but both classes were inactive against bovine trypsin, chymotrypsin and porcine pancreatic elastase at concentrations less than 10(-4)M. The data suggest that drug binding to HLE occurs by stereospecific electrostatic interactions at site(s) removed from the catalytic reaction centre.
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PMID:Synthetic inhibitors of human leukocyte elastase Part 1--Sulphated polysaccharides. 258 75


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