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)

The purpose of this study was to purify and identify the proteinase-like substance previously recognized as responsible for the Na+/K(+)-ATPase stimulating property of plasma from insulin-dependent diabetic subjects. Anion-exchange chromatography followed by two-step heparin affinity chromatography resulted in a fraction highly enriched in both potent Na+/K(+)-ATPase stimulating activity and potent proteolytic activity. Approx. 400 micrograms of purified protein was isolated from 62 mg of starting plasma proteins. When analyzed on sodium dodecyl sulfate gels the active fraction consisted mainly of one polypeptide band with an apparent molecular mass of 66 kDa under either reducing or nonreducing conditions. The proteinase-like properties of the purified fraction were further revealed by its ability to clot plasma, split fibrinogen with production of fibrinopeptide A and induce shape change in human platelets and irreversible platelet aggregation in the presence of the stable analogue of endoperoxides U46619. Its additional capacity to affect platelet phosphoinositol metabolism was shown by the stimulation of protein kinase C-dependent phosphorylation of 47 kDa platelet membrane protein. In designing an identification protocol for the purified fraction, it was postulated that plasma proteinases are probably bound to their inhibitors, to form a stable covalently linked complex. The possibility that a proteinase-proteinase inhibitor complex was purified instead of single proteinase(s) was investigated. Neither trypsin nor neutrophil elastase were present in the active fraction whereas, among the possible plasma proteinase inhibitors tested, immunoreactivity was observed only in the presence of alpha 1-antitrypsin (alpha 1 AT) antiserum. Double immunodiffusion showed that control human alpha 1 AT and the plasma-purified fraction shared common antigens. Furthermore, both isoelectric focusing and amino acid composition analysis showed that the two substances were similar. The results obtained indicate that alpha 1 AT is apparently the only active component of the purified fraction from the plasma of insulin-dependent diabetics, thus suggesting that an altered form of the inhibitor is responsible for the broad range of proteinase-like effects elicited by the plasma-purified fraction.
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PMID:Purification of proteinase-like and Na+/K(+)-ATPase stimulating substance from plasma of insulin-dependent diabetics and its identification as alpha 1-antitrypsin. 131 11

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.
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PMID:Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes. 137 Oct 50

The cell-free haemolymph of the mollusc Octopus vulgaris inhibited the proteolytic activity of the thermolysin against the high-molecular-mass substrate hide powder azure. The purified inhibitor was a glycoprotein composed of two identical 180 kDa disulphide-linked subunits. In addition to the inhibition of the metalloproteinase thermolysin, the protein inhibited the serine proteinases human neutrophil elastase, pig pancreatic elastase, bovine chymotrypsin, bovine trypsin and the cysteine proteinase papain. A fraction of the proteinase-inhibitor complex resisted dissociation after denaturation indicating that some of the proteinase molecules became covalently bound. The nucleophile beta-aminopropionitrile decreased the covalent binding of proteinases to the Octopus vulgaris protein, suggesting that this interaction is mediated by an internal thiol ester; the reactivity and the amino acid sequence flanking the reactive residues of the putative thiol ester were consistent with this hypothesis. Bound trypsin remained active against the low-molecular-mass chromatogenic substrate H-D-Pro-Phe-Arg p-nitroanilide and was protected from inhibition by active-site-directed protein inhibitors of trypsin; however, the bound trypsin was readily inhibited by small synthetic inhibitors. This indicates that the inhibition of proteinases is accomplished by steric hindrance. The proteinase-inhibitory activity of this protein is characteristic of inhibition by mammalian alpha-macroglobulins and the presence of a putative thiol ester suggests that the Octopus vulgaris proteinase inhibitor is a homologue of human alpha 2-macroglobulin.
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PMID:Purification and characterization of an alpha-macroglobulin proteinase inhibitor from the mollusc Octopus vulgaris. 137 44

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.
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PMID:Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors. 138 68

1. The inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase by the hemolymph of 14 insect species in six orders has been investigated. 2. All samples showed great diversity in terms of both total proteinase inhibitory capacity and specificity. 3. The highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata.
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PMID:Serine proteinase inhibitor profiles in the hemolymph of a wide range of insect species. 139 9

alpha 1-Antitrypsin (AAT) is a potent fluid-phase inhibitor of serine proteases. It forms a tightly bound, stoichiometric complex with these enzymes and is inactivated by cleavage within its reactive center. Evidence is here presented, that the 44-residue C-terminal fragment of AAT, termed SPAAT (short peptide from AAT), is found in human tissue, where it is apparently bound to the extracellular matrix (ECM). The identity of SPAAT was established by amino acid sequence analysis through its 40 N-terminal residues. Placental SPAAT inhibits chymotrypsin, human neutrophil elastase (HNE) and pancreatic elastase, but has no effect on trypsin. Unlike AAT, both placental and chemically-synthesized SPAAT are reversible, competitive inhibitors of chymotrypsin with Kl's of 0.92 and 7.5 microM, respectively. Both AAT and placental SPAAT also bind to diisopropyl fluorophosphate (DFP)-treated HNE as well as cathepsin G. SPAAT may therefore play an important role in the protection of ECM proteins, such as elastin, proteoglycans (PG) and/or collagen, from inappropriate attack by serine proteases.
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PMID:Isolation and serine protease inhibitory activity of the 44-residue, C-terminal fragment of alpha 1-antitrypsin from human placenta. 140 56

We have synthesized four guanidinophenyl-substituted protio enol and iodo enol lactones (3-(4-guanidinophenyl)-6-methylidenetetrahydro-2-pyranone (1), 3-(4-guanidinophenyl)-6-(E)-(iodomethylidene)tetrahydro-2-pyran one (2), 4-(4-guanidinophenyl)-6-methylidenetetrahydro-2-pyranone+ ++ (3), and 4-(4-guanidinophenyl)-6-(E)-(iodomethylidene)tetrahydro-2-pyran one (4)) and tested them for inhibitory activity against some trypsin-like enzymes, namely trypsin, urokinase, tissue plasminogen activator (t-PA), plasmin, and thrombin, as well as alpha-chymotrypsin and human neutrophil elastase (HNE). The beta-aryl-substituted protio lactone 3 was a potent alternate substrate inhibitor of trypsin and urokinase. The alpha-aryl-substituted iodo lactone 2 was a permanent inactivator of urokinase, plasmin, t-PA, thrombin, and alpha-chymotrypsin, exhibiting a relatively high specificity for the former two enzymes. In general, these compounds showed a preference for inactivating trypsin-like enzymes over alpha-chymotrypsin and HNE. Also, within the class of trypsin-like enzymes, there was generally good selectivity of inhibition.
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PMID:Guanidinophenyl-substituted enol lactones as selective, mechanism-based inhibitors of trypsin-like serine proteases. 143 18

Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.
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PMID:Effect of conformational mobility and hydrogen-bonding interactions on the selectivity of some guanidinoaryl-substituted mechanism-based inhibitors of trypsin-like serine proteases. 144 34

In the nanomolar enzyme and inhibitor concentration range, 1 mol of mucus proteinase inhibitor (MPI) inhibits 1 mol of neutrophil elastase, cathepsin G, trypsin, and chymotrypsin. In the micromolar concentration range, the enzyme:inhibitor binding stoichiometry is still 1:1 for elastase but shifts to 2:1 for the three other proteinases. These data could be confirmed by three nonenzymatic methods: (i) fluorescence anisotropy measurements of mixtures of proteinases with 5-dimethylaminonaphthalene-1-sulfonylated or fluoresceinylated MPI, (ii) absorption spectrocospy of fluorescein-MPI-proteinase complexes isolated by gel filtration, (iii) analytical ultracentrifugation which showed that the molecular mass of the MPI-chymotrypsin complex is 56 kDa, whereas that of the MPI-elastase complex is 39 kDa. The binary MPI-elastase complex is unable to inhibit trypsin or cathepsin G. On the other hand, 1 mol of elastase displaces 2 mol of trypsin or cathepsin G from their ternary complexes with MPI.
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PMID:The proteinase: mucus proteinase inhibitor binding stoichiometry. 153 27


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