Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A chimeric plasminogen activator, GPRP-u-PA (144-411), consisting of the Gly-Pro-Arg-Pro tetrapeptide fused to the N-terminal of a truncated urokinase-type plasminogen activator (comprising Leu 144 through Leu 411), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. After renaturation, the chimera was purified to homogeneity with specific amidolytic activity of 100,000 IU/mg protein. The chimera showed 6-fold greater affinity for fibrin clots than native low molecular weight urokinase (LUK) and 1.5-fold greater affinity than a chemical conjugate, GPRP-LUK, generating via coupling Gly-Pro-Arg-Pro tetrapeptide to native low molecular weight urokinase. The chimera had 2 to 3 fold greater fibrinolytic potency than native LUK in vitro. Fibrinogen had no influence on fibrinolysis of the chimera. The chimera consumed much less fibrinogen than native LUK.
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PMID:Characterization of a recombinant chimeric plasminogen activator composed of Gly-Pro-Arg-Pro tetrapeptide and truncated urokinase-type plasminogen activator expressed in Escherichia coli. 867 Feb 47

A method for the localization of activities of proteases using substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group is described. 0.1 ml of 5-20 mMol solution of the respective substrate (Gly-Pro-AFC, Ala-Pro-AFC, Z-Ala-Arg-Arg-AFC, Z-Gly-Arg-Arg-AFC, Z-Gly-Gly-Arg-AFC, D-Val-Leu-Lys-AFC) in dimethylsulfoxide or dimethylformamide was added to 0.9 ml of 0.1 M Tris-HCl buffer, pH 7.4-7.8 or 0.1 M cacodylate buffer, pH 5-5.5. In the case of Z-Ala-Arg-Arg-AFC (cathepsin B substrate) 15 mM EDTA and 12 mM dithiothreitol were added. 7 mM amiloride or 2 mg/1 ml aprotinin were used as inhibitors with Z-Gly-Gly-Arg-AFC (urokinase substrate) and with D-Val-Leu-Lys-AFC (plasmin substrate). Substrate solutions were mixed with an equal amount of 2% agar solution in distilled water or in the respective buffer the pH of which was adjusted according to the pH optimum of the enzyme to be demonstrated. The agar solution was kept in a water bath at a temperature of 50-60 degrees C. After careful mixing, the substrate solution in agar was poured into a cylindrical vessel closed with a semipermeable membrane (Nephrophan) on which unfixed cryostat sections were mounted. 1-5 mM AFC solution in dimethylsulfoxide or dimethylformamide instead of the substrate was used as the control. Quenched samples of rat kidney and jejunum, biopsies of human jejunal mucosa, and of colorectal and uterine tumors were employed for the preparation of sections. After gelification of the medium in a refrigerator the vessels with sections were incubated in the dark at 37 degrees C for 0.5-several h. The reaction was controlled in a fluorescence microscope with an epiillumination adjusted to the FITC fluorescence and documented. A yellowish green fluorescence depicts sites where AFC was set free (sites with enzyme activity). When the reaction reached the required intensity the membranes were cut off, transferred to glass slides, mounted in glycerol, observed and photographed immediately (due to the solubility of AFC in glycerol). An acceptable cellular localization was achieved. The method with AFC substrates can be recommended for comparative biochemical and histochemical studies of proteases using the same substrate and for cases in which no other reliable procedure for the localization of the respective enzyme activity is available (e.g. urokinase, plasmin).
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PMID:The use of substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group in the localization of protease activities in situ. 873 6

Two low molecular weight urokinase derivatives were obtained by covalent coupling of a synthetic Gly-Pro-Arg-Pro tetrapeptide to peptide A of low molecular weight urokinase and exchanging the native peptide A of low molecular weight urokinase with Gly-Pro-Arg-Pro-peptide A to obtain derivative I or direct conjugation of Gly-Pro-Arg-Pro tetrapeptide to low molecular weight urokinase to obtain derivative II. In caseinolytic assay, fibrin can stimulate the two derivatives to activate plasminogen. But the two derivatives showed different kinetic behaviors. The derivative I displayed immediate onset of lysis and derivative II displayed a lag phase.
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PMID:Chemical conjugation of Gly-Pro-Arg-Pro. Tetrapeptide to low molecular weight urokinase. 882 96

A Gly-Pro-Arg-Pro tetrapeptide, homologous to amino-terminal segment of the human fibrin alpha chain after the release of the fibrinopeptide A, was covalently coupled to peptide A of low molecular weight urokinase. The resulting derivative gained increased affinity for fibrin. In caseinolytic assay, fibrin can stimulate the derivative to activate plasminogen. The derivative had two-fold greater fibrinolytic potency than native low molecular weight urokinase and its affinity for fibrin clot was 3.9-fold higher than that of low molecular weight urokinase.
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PMID:A low molecular weight urokinase derivative with enhanced fibrin affinity. 884 49

CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the coboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg-->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54-Asn57). The alteration 55Arg-->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr-->Gly and 61Tyr-->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr-->Gly mutation was non-functional. The mutation of 61Tyr-->His diminished function but changes 61Tyr-->Ala and 61Tyr-->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.
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PMID:Structure-function relationships of the complement regulatory protein, CD59. 907 80

We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.
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PMID:Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase. 916 91

The in-vitro effects of citrus, red and white wines on human plasma fibrinolytic enzymes were compared. When citrus wine was added to plasmin, H-D-Val-Leu-Lys-pNA (S-2251) amidolysis was not changed. Although, it was significantly inhibited when red and white wines were added. The pyro-Glu-Gly-Arg-pNA (S-2444) amidolysis of the plasminogen activator urokinase was inhibited by all types of wine. Since the same amount of Et-OH as the wine's content did not effect on these inhibitions, it can be assumed to be due to other unknown substances in wines besides alcohol. In in-vivo test of 20 normal volunteers (ages 20-52) with various wines equivalent to 30-60 ml of alcohol content, the coagulation parameters of gamma and kappa values on thromboelastography were not so much changed after 1-2 hrs of the application. Also, there was not much difference of the fibrinolytic parameters in terms of the plasma euglobulin clotlysis time, S-2444 amidolysis, and Ma values on thromboelastography. However, the protein concentration and the enzyme activity of the urokinase-like plasminogen activator, which were extracted from the plasma of volunteers who had the citrus wine 1 hr before the test were higher than twice of the before control. The main molecular form of this enzyme was proved to have a molecular weight of about 30,000 by zymography. It is concluded that not only wines induce the endogenous plasminogen activator just like other alcoholic beverages but also wines contain a fibrinolysis inhibitor which works directly to the enzyme dissolubility. This finding warns that extremely complex results can be expected depending on the test method.
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PMID:Effects of wine on plasma fibrinolytic and coagulation systems. 970 4

The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val-->Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80+/-9% (mean+/-S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 86+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degreesC, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52+/-5.2%, 55+/-8.2% and 46+/-4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86+/-1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.
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PMID:Characterization of plasminogen activator inhibitor 1 mutants containing the P13 to P10 region of ovalbumin or antithrombin III: evidence that the P13 residue contributes significantly to the active to substrate transition. 974 34

Membrane binding of urokinase type plasminogen activator (u-PA) is thought to play a pivotal role in connective tissue remodeling and invasive processes. We compare the ability of different matrix-metalloproteinases involved in connective tissue turnover to cleave pro-urokinase type plasminogen activator between the catalytic domain and the receptor binding part to investigate a potential role for matrix-metalloproteinases in the regulation of membrane-associated proteolytic activity. We employed several forms of human stromelysin-1 (full length, C-truncated, and recombinant catalytic domain), rabbit C-truncated stromelysin-1, the human gelatinases A and B and the human catalytic domain of neutrophil collagenase. The gelatinases and the collagenase did not separate the receptor binding domain of pro-urokinase type plasminogen activator from the catalytic domain, whereas all stromelysin-1 forms cleaved the glutamic acid 143-leucine 144 bond of pro-urokinase type plasminogen activator. This reaction could be inhibited by specific inhibitors of matrix metalloproteinases and was not affected by inhibitors of serine proteinases. The M(r) 31000 cleavage product with leucine 144 as N-terminus displayed no proteolytic activity towards the pro-urokinase type plasminogen activator substrate pyroGlu-Gly-Arg-pNA-HCI (S2444), but it could be activated by an additional treatment with plasmin. Comparison between full length stromelysin-1 and its C-truncated forms, showed that both exhibited the same cleavage properties towards pro-urokinase type plasminogen activator. Thus, the cleavage of pro-urokinase type plasminogen activator by stromelysin-1 is not influenced by the presence or absence of the C-terminal domain. The recombinant catalytic domain of MMP-3 generated pro-urokinase type plasminogen activator, whereas incubation of pro-urokinase type plasminogen activator with the native forms of human or rabbit stromelysin-1 led to a moderate activation of pro-uPA due to an additional cleavage that is catalyzed by a serine proteinase.
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PMID:The cleavage of pro-urokinase type plasminogen activator by stromelysin-1. 980 93

This is the first study in which u-PA activity is detected in situ during SCL wear. The histochemical localization of u-PA activity is done by the methods of Lojda using unfixed cryostat sections on semipermeable membranes and a gel incubation medium containing sensitive substrates with the 7-amino-4-trifluoromethylcoumarin (AFC) (Enzyme Systems Products, Sierra Lane, Dublin, CA, USA) leaving groups. Z-Gly-Gly-Arg-AFC and Glut-Gly-Arg-AFC were employed as the substrates. The results show that in the normal,cornea u-PA activity is absent. Also the wearing of SCL does not evoke the appearance of u-PA activity in the cornea within the first three days. On day 4 the first u-PA activity appears; it is located in the superficial layers of the corneal epithelium. On day 7 of SCL wear, u-PA activity is present in all layers of the corneal epithelium and (to a lesser extent) also in the comeal endothelium; keratocytes of the corneal stroma are only slightly active for u-PA. Extended SCL wear (for two weeks) leads to an increase of u-PA activity in keratocytes beneath the epithelium. Also, some inflammatory cells (mainly polymorphonuclear leukocytes, PMNs) present in the corneal stroma are enzymatically active. After three weeks of SCL wear the number of PMNs in the corneal stroma increases; some PMNs are highly active for u-PA. In the corneal endothelium the u-PA activity is also highly pronounced. It can be concluded that extended SCL wear leads to the gradual increase of u-PA activity in the rabbit cornea. It is suggested that active u-PA is involved in the corneal damage related to SCL wear.
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PMID:The appearance and possible role of plasminogen activator of urokinase type (u-PA) activity in the cornea related to soft contact lens wear in rabbits. 1043


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