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

Central venous catheters are being used with increasing frequency to administer drugs, and as a result, catheter obstruction caused by precipitation of poorly soluble fluid components has become a common problem. We report our first experience using 0.1 N hydrochloric acid to restore patency to central venous catheters obstructed from insolubility-induced precipitation. Precipitation was caused by drug as well as calcium and phosphorus incompatibilities. The initial use of urokinase in two cases was unsuccessful in restoring catheter patency. In all four cases, the instillation of 0.2-1.0 ml of HCl cleared the catheters. Catheter patency usually was gained immediately. No side effects were noted. Our experience supports preliminary data (JPEN 9 (suppl):255, 1985) which suggest that 0.1 N HCl is effective in clearing insolubility-induced precipitation in central venous catheters.
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PMID:Use of hydrochloric acid to clear obstructed central venous catheters. 318 23

Urokinase (UK) was immobilized onto the inner surface of 4-mm ID canine autogenous fibrocollagenous tubes in order to develop a fibrinolytic small-caliber vascular prosthesis. Using a glutaraldehyde entrapment process, the immobilized urokinase was compared to its soluble counterpart for its ability to degrade fibrin. Fibrin Degradation Product (FDP) levels increased with time for both soluble and immobilized urokinase, where FDP levels at complete lysis for both soluble and immobilized urokinase, where FDP levels at complete lysis were measured at 165.83 +/- 6.44 micrograms/ml. The rate of fibrinolysis was slower for the immobilized urokinase compared to its soluble form. The inhibitory effect of alpha-2 plasmin inhibitor was shown to be inimical to urokinase activity. Using a differential recirculation reactor apparatus and an esterolytic reaction involving N-alpha-acetyl-L-lysine methyl ester HCl, the amount of urokinase immobilized onto fibrocollagenous tubes (FCT) was measured to be 110 +/- 14 CTA units of UK per millimeter graft. The stability of immobilized urokinase was maintained for at least 75 hours of operation time, which corresponds to 1 year maintained for at least 75 hours of operation time, which corresponds to 1 year of storage time. Urokinase-bound fibrocollagenous tubes (UK-FCT), in addition to control FCT's, Perloff prostheses, and the autogenous saphenous vein, were interposed onto the carotid artery of canine mongrels using end-to-end anastomoses with 7-0 prolene sutures. Results indicate that the patency rates for UK-FCT's were 86% and 62% after 1 and 2 months, respectively. The Perloff graft and the control FCT had much lower patency rates. These results indicate that immobilizing urokinase is a viable method of producing a small-caliber biocompatible vascular prosthesis.
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PMID:Fibrinolytic activity of immobilized plasminogen activator. 405 55

This study demonstrates that human plasma alpha(2)-macroglobulin preparations possess an enzymic activity that degrades fibrinogen, resulting in the formation of products whose structure resembles that of circulating fibrinogen catabolites. The sequence of degradation is similar to that observed in plasmin-catalyzed digests, in that Aalpha-chain fragmentation precedes that of Bbeta-chain. The addition of plasminogen activators to plasma induced an increase in the N-alpha-tosyl-l-arginine methyl ester HCl esterase and fibrinogenolytic activity associated with alpha(2)-macroglobulin purified from this plasma, indicating that the enzymic activity of the complex was preserved and could be increased in the presence of other plasma enzyme inhibitors. Immunochemical studies demonstrated that an alpha(2)-macroglobulin-plasmin complex had formed in urokinase-treated plasma. This alpha(2)-macroglobulin preparation manifested an esterolytic profile like that of a complex prepared from plasmin and purified alpha(2)-macroglobulin. After complex formation with alpha(2)-macroglobulin in plasma, plasmin retained less than 0.1% of its fibrinogenolytic activity. That plasmin expressed its activity while bound to alpha(2)-macroglobulin was suggested by immunoprecipitation of this activity with alpha(2)-macroglobulin antibody and by the demonstration that pancreatic trypsin inhibitor did not effectively inhibit its fibrinogenolytic or esterolytic activity. These results raise the possibility that, in addition to its activity as a major plasma proteolytic enzyme inhibitor, alpha(2)-macroglobulin may modulate enzyme-substrate interactions, such as those resulting in the formation of circulating fibrinogen catabolites, by providing a mechanism for the preservation and protection of a portion of the enzymic activity in the presence of other circulating inhibitors.
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PMID:Degradation of human fibrinogen by plasms alpha2-macroglobulin-enzyme complexes. 426 29

A single SH group in the B chain (33 kDa), generated by the specific reduction of the single interchain SS bond of human urinary urokinase, was alkylated (UK X B) with iodoacetamide to prevent a spontaneous SH-SS interchange. An SS bond in UK X B was exclusively alkylated with iodoacetamide (R X CAM-UK X B) after reduction with dithiothreitol in 0.3 M guanidine X HCl in the presence of the competitive inhibitor N alpha-benzoyl-L- argininamide with concomitant loss of 65-68% of the esterolytic activity towards N-acetyl-glycyl-L-lysine methyl ester. This specific SS bond was located at Cys194 - Cys222 whose SS loop contained the active-site Ser198 , as determined by amino acid analyses and identification of the N and C termini of the tryptic digest. Transformation of UK X B into R X CAM-UK X B induced no shift of the optimal pH in the bell-shaped pH/activity profile; pH values for 50% activity were similar (pH 9.7) for 10-min alkalization of the enzyme but different between UK X B (pH 9.4) and R X CAM-UK X B (pH 8.8) for 18-h alkalization. An unaltered Km value and a decline by 64% in kcat in the esterolytic activity indicate that the pretransition Michaelis complex is formed without degeneration of the primary substrate-binding site, but the catalytic pathway thereafter has deteriorated. In affinity labeling with dansyl chloride or N alpha-tosyl-L-lysine chloromethylketone, which interrupted the catalysis at the latest at a stage involving the abortive acyl intermediate, the second-order rate constant for UK X B was lowered to 28% or 35% for R X CAM-UK X B, respectively, but the labeling yields were similar. The results indicate that indispensable structural elements, such as the catalytic triad and oxyanion hole, are maintained but a local conformation, which is necessary for efficient transition to the acyl intermediate and/or for resistance against alkaline inactivation, is destabilized with Cys194 - Cys222 scission.
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PMID:A specific disulfide bond associated with the activity of human urokinase. Its topological identification and reductive cleavage followed by kinetic changes in enzymatic reaction and affinity labeling. 672 47

The kinetics of urokinase hydrolysis of the low molecular weight substrates--S-2444 (Glu-Gly-Arg-pNA.HCl), N-benzoyl-L-arginine ethyl ester and acetylglycyllysine methyl ester have been studied. The kinetic constants for the hydrolysis reactions have been determined and the appropriate schemes have been proposed. The native enzyme is shown to be highly thermostable.
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PMID:[Kinetics of urokinase hydrolysis of low molecular weight substrates]. 704 94

A simple rapid fluorescent method for the detection of plasminogen activator activity of urokinase type (u-PA) in the tear fluid is described. Small filter paper punches were soaked in the substrate solution (Z-Gly-Gly-Arg-trifluoromethylcoumarinyl-7-amide, 1 mg/1 ml) and aprotinin 100 micrograms/1 ml) dissolved in 0.1 M Tris-HCl buffer, pH 7.2 and dried. The dried punches were soaked with tears (by direct contact of the punch with the site where the activity should be assessed or by dropping of 3-5 microliters of tears collected by a glass micropipette). The punches were incubated in a thermostat (37 degrees C) together with punches containing a known u-PA activity (calibrated punches) in preheated (37 degrees C) Petri dishes. In 1 min intervals (during the first 15 min) and in 5 min intervals thereafter the probes were exposed to UV light, and the time of the first appearance of a bright yellow fluorescence was recorded. In punches containing 5 IU u-PA activity fluorescence appeared after 2 min incubation; 2.5 IU were detected after 5 min, 1.25 IU after 15 min, 0.625 IU after 30 min, 0.313 IU after 60 min, 0.156 IU after 90 min, and 0.078 IU after 120 min incubation. This simple method is recommended for use particularly in clinical laboratories. It enables e.g. to obtain a rather quick information about the urokinase activity in the tear fluid and to start the treatment with an appropriate inhibitor, if necessary.
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PMID:Biochemical and histochemical studies of plasminogen activator of urokinase type (u-PA) activity. I. A simple rapid semiquantitative fluorescent method for its detection in the tear fluid. 751 Sep 19

The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).
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PMID:Role of activated protein C in facilitating basement membrane invasion by tumor cells. 826 50

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

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.
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PMID:Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. 926 Nov 79

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.
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PMID:Proteases with plasminogen activator activity in hamster oviduct. 1060 73


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