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)

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.
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PMID:Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes. 776 51

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.
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PMID:COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization. 785 21

An expression system for alpha 1-antitrypsin in Escherichia coli was developed using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polymerase gene resulted in about 30% of newly synthesized protein being alpha 1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha 1-antitrypsin on its activity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation of Ala347 to threonine in alpha 1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of this mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha 1-antitrypsin. Cleavage within the reactive-site loop of wild-type alpha 1-antitrypsin causes a conformational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha 1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of mutations in the hinge region of serpins. 834 75

Activities of plasmin and trypsin contained in complexes with alpha 2-macroglobulin, related to pregnancy alpha 2-glycoprotein and with protein A were studied in presence of various inhibitors with a molecular mass of 0.2-70.0 kDa. Specific characteristics of interaction between plasmin and these macroglobulins inhibitors as well as with alpha 1-inhibitor of proteinases, antithrombin III and alpha 2-antiplasmin were studied using electrophoresis and immunoblotting.
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PMID:[Study of structural features of macroglobulins complexed with plasmin]. 837 18

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.
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PMID:Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line. 840 7

The wealth of structural information now available for thrombin, its precursors, its substrates, and its inhibitors allows a rationalization of its many roles. alpha-thrombin is a rather rigid molecule, binding to its target molecules with little conformational change. Comparison of alpha-thrombin with related trypsin-like serine proteinases reveals an unusually deep and narrow active site cleft, formed by loop insertions characteristic of thrombin. This canyon structure is one of the prime causes for the narrow specificity of thrombin. The observed modularity of thrombin allows a diversity in this specificity; its "mix-and-match" nature is exemplified by its interactions with macromolecules (Fig. 20). The apposition of the active site to a hydrophobic pocket (the apolar binding site) on one side and a basic patch (the fibrinogen recognition exosite) on the other allows for a fine tuning of enzymatic activity, as seen for fibrinogen. Thrombin receptor appears to use the same sites, but in a different way. Protein C seems only able to interact with thrombin if the recognition exosite is occupied by thrombomodulin. These two sites are also optimally used by hirudin, allowing the very tight binding observed; thrombin inhibition is effected by blocking access to the active site. On the other hand, antithrombin III makes little use of the recognition exosite; instead, its interactions are tightened with the help of heparin, which binds to a second basic site (the heparin binding site). Thrombin's modularity is a result of the conjunction of amino acid residues of like properties, such as charge or hydrophobicity. The charge distribution plays a role, not only in the binding of oppositely charged moieties of interacting molecules, but also in selection and preorientation of them. Nonproteolytic cellular properties are attributed to 1) the rigid insertion loop at Tyr60A, and 2) a partially inaccessible RGD sequence. The former can interact with cells in the native form; the latter would appear to be presented only in an (at least partially) unfolded state. The membrane binding properties of prothrombin can be understood from the ordered arrangement of calcium ions on binding to the Gla domain. Kringle F2 binds to thrombin at the heparin binding site through charge complementarity; a conformational change appears to occur on binding. The observed rigidity of the thrombin molecule in its complexes makes thrombin ideal for structure based drug design. Thrombin can be inhibited either at the active site or at the fibrinogen recognition exosite, or both.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A player of many parts: the spotlight falls on thrombin's structure. 846 68

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.
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PMID:Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III. 848 44

The inhibition of proteinase activity by members of the serine proteinase inhibitor (serpin) family is a critical regulatory mechanism for a variety of biological processes. Once formed, the serpin enzyme complexes (SECs) are removed from the circulation by a hepatic receptor. The present study suggests that this receptor is very likely the low density lipoprotein receptor-related protein (LRP), a prominent liver receptor. In vitro binding studies revealed that antithrombin III (ATIII)-thrombin, heparin cofactor II (HCII)-thrombin, and alpha1-antitrypsin (alpha1AT)-trypsin bound to purified LRP, and their binding was inhibited by the 39-kDa receptor-associated protein (RAP), an antagonist of LRP-ligand binding activity. In contrast, native or modified forms of the inhibitors were unable to bind to LRP. Mouse embryonic fibroblasts, which express LRP, mediate the cellular internalization leading to degradation of these SECs, while mouse fibroblasts genetically deficient in LRP showed no capacity to internalize and degrade these complexes. SECs were also degraded by HepG2 cells, and this process was inhibited by LRP antibodies, RAP, and chloroquine. The cellular-mediated uptake and degradation was specific for SECs; native or modified forms of the inhibitors were not internalized and degraded. Finally, in vivo clearance studies in rats demonstrated that RAP inhibited the clearance of ATIII-125I-thrombin complexes from the circulation. Together, these results indicate that LRP functions as a liver receptor responsible for the plasma clearance of SECs.
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PMID:Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and alpha 1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein. 862 56

Tryptase (EC 3.4.21.59), the major secretory product of human mast cells, has become widely used as a biochemical marker for mast cells and mast cell activation, and is attracting attention as a mediator of allergic disease. However, there is little information available on the properties, or even the presence, of this protease in commonly used species of laboratory animals. We, here, report the demonstration and characterisation of this enzyme in the guinea pig lung. Tryptic activity resistant to alpha 1-proteinase inhibitor and soybean trypsin inhibitor was detected in sections of guinea pig lung tissue with the histochemical substrate Z-Gly-Pro-Arg-MNA. It was localised to mast cells and appeared to be present in all mast cells staining with Alcian Blue. A tryptic protease was purified 2400-fold from whole lung tissue by high salt extraction, cetylpyridinium chloride precipitation, heparin agarose chromatography, and gel filtration. This enzyme was found to be multimeric with a subunit of 38 kDa and a native molecular mass of 860 +/- 100 kDa. Inhibitor studies identified it as a serine protease. Like human tryptase, it was inhibited by leupeptin, benzamidine, and APC 366 (N-(1-hydroxy-2- naphthoyl)-L-arginyl(-L-prolinamide hydrochloride), but not by alpha 1-proteinase inhibitor, soybean trypsin inhibitor, or antithrombin III. Its response to changes in pH and ionic strength was similar to that of human tryptase. Differences between the guinea pig and human enzymes were seen in activity toward a panel fo 10 tryptic p_nitroanilide peptide substrates. Kinetic constants were determined for two of these: with L-Pyr-Pro-Arg-pNA the guinea pig tryptase had a similar Km but a 5-fold lower kcat than human tryptase, and with L-Pyr-Gly-Arg-pNA the guinea pig enzyme had a 10-fold lower Km and a 30% greater kcat than human counterpart. Heparin stabilised guinea pig tryptase, but did not alter its kinetic parameters as it did with human tryptase, decreasing the Km towards both substrates. The presence of a protease with similarities to human tryptase in the mast cells of guinea pigs suggests that this species may be an appropriate model to investigate the actions to tryptase in vivo, provided cognizance is taken of the differences that do exist.
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PMID:Guinea pig lung tryptase. Localisation to mast cells and characterisation of the partially purified enzyme. 869 58

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.
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PMID:Inhibition of coagulation factors by recombinant barley serpin BSZx. 884 56


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