Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P35237 (thrombin inhibitor)
 2,012 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The jawed leech, Hirudinaria manillensis is closely related to Hirudo medicinalis, both belonging to the same family Arhynchobdellida. From Hirudo, two potent peptide inhibitors, hirudin (a thrombin inhibitor) and eglin (an elastase/chymotrypsin inhibitor) have been characterised in detail. During our studies to isolate thrombin inhibitor from the leech Hirudinaria a potent inhibitor, analogous to eglin, was also detected. Results indicate that this inhibitor, which we have named 'GELIN', is significantly different from eglin. Gelin was isolated and purified to homogeneity by ion exchange chromatography and reverse phase HPLC. The isoelectric point of Gelin was estimated to be 4.55, in contrast to 6.45 for eglin. The molecular weight of Gelin was similar to eglin, as estimated by SDS-PAGE. Amino-terminal sequence analysis of the first 29 residues show no sequence homology with eglin or any other serine protease inhibitors. Circular dichroism studies showed that the secondary structure of Gelin has no helix, 58% beta sheets and 42% random structures compared to 19% helix, 56% beta sheets and 25% random structures in eglin. Like eglin, Gelin inhibits elastase, cathepsin G and chymotrypsin but has little or no activity towards plasmin, thrombin, pepsin and trypsin. These data suggest that the elastase inhibitors from these two species of leech are fundamentally different in structure, indicative of independent evolutionary origin.
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PMID:Biochemical characterisation of a pancreatic elastase inhibitor from the leech Hirudinaria manillensis. 128 66

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. 130 49

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.
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PMID:Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin. 201 64

We have recently reported that exogenous thrombin produced a dose- and endothelium-dependent coronary vasodilation in both intact open-chested dogs and in isolated dog coronary artery preparations. To determine whether the observed vasodilatory effect may be related to thrombin proteolytic enzymatic activity, effects of other proteases, such as trypsin, chymotrypsin, and pepsin, on the mechanical responses of isolated dog coronary arteries were studied. Among the four proteases evaluated, only thrombin (0.01-0.1 U/ml) and trypsin (0.03-0.67 U/ml) consistently produced a potent dose- and endothelium-dependent relaxation, that was reproducible with repeated testings. Addition of chymotrypsin (0.01-1.0 U/ml) produced only a minimal effect and was not reproducible, while addition of pepsin, as much as 10 U/ml, did not produce any effect. The specific soybean trypsin inhibitor and aprotinin, but not heparin and hirudin, competitively shifted the trypsin dose-response to the right, whereas heparin, hirudin, and antithrombin III proved to be more effective than trypsin inhibitors in inhibiting the thrombin-induced vasodilation. In all cases, the thrombin- and trypsin-induced vasodilation were equally sensitive to inhibition by the specific synthetic thrombin inhibitor, PPACK (D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone, 1-30 nM). PPACK, however, had no effect on the other endothelium-dependent coronary vasodilators, such as acetylcholine and adenosine triphosphate, in our isolated dog coronary artery preparations. Biochemical determinations of the amidolytic activity of thrombin, using Tosylglycyl-L-prolyl-L-arginine-p-nitroanilide as a chromogen, also indicated a similar PPACK and heparin-antithrombin III dose-dependent inhibition of the thrombin enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of thrombin-induced endothelium-dependent coronary vasodilation in dogs: role of its proteolytic enzymatic activity. 241 89

We compared lung fibroblast growth-stimulating activity (FGA) of several serine proteases including thrombin in vitro, and examined the mechanism of FGA. FGA was measured by incorporation of 3H-thymidine into lung fibroblasts (IMR-90). The activities of the enzymes were measured by spectrofluorometric method with synthetic peptides specific for each enzyme, and these enzymes were added to the assay system for FGA at concentrations of 7 x 10(0)-7 x 10(5) unit/ml. Human thrombin, bovine trypsin and bovine alpha-chymotrypsin showed clear FGA, but that of alpha-chymotrypsin was lower than those of thrombin and trypsin. On the other hand, porcine pancreatic elastase and human neutrophil elastase did not show any FGA, and had a cytotoxic effect on fibroblasts. A specific low molecular-weight thrombin inhibitor, argatroban (MW. 562), inhibited not only the enzyme (peptidolytic) activity of thrombin, but also its FGA at the same concentration. These results suggest that serine proteases can be classified into at least two groups, showing FGA and cytotoxic activity, respectively, and that the FGA of the former group is mediated by their catalytic (enzymatic) action.
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PMID:[Lung fibroblast growth-stimulating activity of serine protease]. 827 61

Human cytoplasmic antiproteinase (CAP) is an intracellular serpin that has been reported to utilize Arg341 as the reactive site P1 residue to neutralize a broad variety of extracellular serine proteases with trypsin-like specificity. Both native CAP and recombinant CAP purified from Escherichia coli were observed to form SDS-stable complexes not only with 125I-thrombin and 125I-urokinase, but also with 125I-chymotrypsin. Kinetic studies indicated that the amidolytic activity of chymotrypsin is inhibited efficiently and rapidly by CAP in a two-step process with a dissociation constant Ki of an initial loose complex of 3.3 nM, a forward isomerization rate constant k2 to the tight complex of 0.014 s-1, and an overall second order association rate constant of 6 x 10(6) M-1 s-1, similar to the kinetic constants obtained for the formation of the trypsin-CAP complex. N-terminal amino acid sequencing and mass spectrometry indicated that chymotrypsin interacts with CAP at Met340, in contrast to thrombin, which interacts as expected at Arg341. Thus, CAP is the first serpin that has been shown to be capable to inhibit efficiently and with similar association rate constants different proteases at distinct reactive site residues, strongly supporting the notion of a highly mobile and flexible serpin reactive site loop and suggesting that this inhibitor may have evolved separate reactive sites for the specific regulation of different proteolytic activities.
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PMID:Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteases utilizing distinct reactive site residues. 866 39

A cDNA encoding of the serine proteinase inhibitor (serpin), B-43, was cloned from the cDNA library of the bovine brain. It encoded 378 amino acids, and the MW of the protein was estimated to be 42.6 kDa, which is consistent with that of the native B-43 purified from the bovine brain. The homology search revealed that B-43 belongs to the ovalbumin branch of the serpin superfamily. Among them, B-43 was most homologous to human placental thrombin inhibitor (PI-6) and its murine counterpart, with the amino acid identity of 76% and 71%, respectively. Northern blot analysis showed that the size of the transcript was 1.4 kb, and that the expression of B-43 in the bovine brain varied depending on the brain regions, i.e. a lower level of expression was observed in the cerebral cortex and the hippocampus compared to the level of expression that was observed in the medulla oblongata. [35S]-labeled B-43 protein was synthesized in vitro by using a rabbit reticulocyte lysate system, which formed complexes with proteinases such as thrombin, trypsin, alpha-chymotrypsin, and 7S nerve growth factor (NGF), but not with urokinase or plasmin. These results, together with the immunohistochemical localization of B-43 in astrocytes and in some neurons which was observed in the previous study suggest that B-43 may be involved in the regulation of serine proteinases present in the brain or extravasated from the blood.
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PMID:Cloning of a serine proteinase inhibitor from bovine brain: expression in the brain and characterization of its target proteinases. 901 86

Proteinase inhibitor 6 (PI-6) is a 42-kDa intracellular protein present in epithelial cells and endothelial cells. It is capable of inhibiting a number of serine proteinases, including trypsin and chymotrypsin. In this study we examined PI-6 expression in human skin and its primary cell type, the keratinocyte. By immunohistochemical analysis, PI-6 staining is absent from the basal cells, weak in the spinous layer, and strongest in the granulosa layer of human epidermis. Immunoblotting of cultured primary keratinocytes revealed that PI-6 production increases 24-fold on differentiation. Analysis of an immortalized keratinocyte cell line, HaCat, showed a 5-fold increase in PI-6 mRNA and a 7-fold increase in PI-6 protein upon differentiation, and indirect immunofluorescence revealed that this is due to an increase in the number of differentiated cells expressing high levels of PI-6. Of particular interest is the appearance of a preformed complex between PI-6 and an endogenous serine proteinase in differentiating HaCat cells, which was detected by a monoclonal antibody demonstrated to preferentially recognize PI-6 in complex with a proteinase. This identification of a PI-6/proteinase complex is the first example of a serpin bound to a proteinase in keratinocytes. We postulate that a physiological role of PI-6 is to regulate a serine proteinase associated with keratinocyte differentiation.
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PMID:Proteinase inhibitor 6 (PI-6) expression in human skin: induction of PI-6 and a PI-6/proteinase complex during keratinocyte differentiation. 985 66

Synthesis of a variety of 5,5-trans fused lactones, related to compounds found in extracts of Lantana camara, has provided a series of novel acylating inhibitors of human thrombin, trypsin, chymotrypsin and human leucocyte elastase. The most effective thrombin inhibitor is 7 with an IC50 of 130 nM and a Kobs/[1] of 4,000 M-1 s-1.
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PMID:5,5-trans lactone-containing inhibitors of serine proteases: identification of a novel, acylating thrombin inhibitor. 987 54

From the bloodsucking bug Dipetalogaster maximus, a protein with anticoagulant activity was isolated and biochemically characterized. The isolated protein, named dipetalogastin, possesses an average molecular mass of 11.8 kD. Its N-terminal sequence shows homology to rhodniin, a thrombin inhibitor isolated from the bug Rhodnius prolixus. The in vitro anticoagulant activity of dipetalogastin occurs via the inhibition of thrombin. The anticoagulant and thrombin inhibitory potency of dipetalogastin is comparable to that of recombinant hirudin. Its specific thrombin inhibitory activity is 9,300 antithrombin units/mg protein. Dipetalogastin forms only 1:1 molar complexes with thrombin. It is a tight-binding inhibitor of thrombin possessing a dissociation constant of 125 fM. It does not inhibit factor Xa or alpha-chymotrypsin and only weakly inhibits trypsin.
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PMID:Biochemical characterization of a thrombin inhibitor from the bloodsucking bug Dipetalogaster maximus. 1070 1


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