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)

Changes in plasminogen activator (PA) and PA inhibitor (PAI) activities were measured during follicular development in granulosa cells (GC) and theca tissue (TT) isolated from the six largest yolk-filled preovulatory follicles (F1, F2, F3, F4, F5, F6) and large white follicles (LWF) of the domestic hen. PA activity increased and PAI activity decreased during follicular development, with the peak PA value and minimum activity for PAI observed in the largest preovulatory follicle (F1) 12-14 h before expected time of ovulation. The PA activity in GC and TT appears to be principally of the tissue (t)-PA type judging from its substrate specificity and biochemical characteristics. The enzyme cleaved the chromogenic substrate specific for t-PA (Spectrozyme TM t-PA; CH3SO2-D-CHT-Gly-Arg-p-nitroanilide) more efficiently (4-6 x) than that for u-PA (Spectrozyme TM UK; Cbo-L-Glu-(alpha-t-BuO)-Gly-Arg-p-nitroanilide), suggesting that t-PA may be the predominant PA in the chicken preovulatory follicle. Determination of PA activity following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focussing suggested the presence of two forms of the enzyme in GC and TT. The predominant form of PA had a molecular weight of 75,000 and an isoelectric point (pI) of 7.7, characteristics similar to those reported for t-PA in humans, pigs, and rodents. The other form of PA had a molecular weight of 35,000 and pI of 8.4. PAI present in GC and TT had a molecular weight of 50,000 and pI of 4.7. In GC, an acid-labile PAI was detected with biochemical characteristics similar to those of the protease, nexin I.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Changes in tissue-type plasminogen activator-like and plasminogen activator inhibitor activities in granulosa and theca layers during ovarian follicle development in the domestic hen. 211 20

Recently, we demonstrated that tissue plasminogen activator directly releases fibrinopeptides A and B (FPA and FPB) from fibrinogen. The purpose of this study was to determine whether urokinase has similar activity. Incubation of urokinase with fibrinogen or heparinized plasma results in concentration-dependent FPB release unaccompanied by FPA cleavage. For equivalent amidolytic activity, high molecular weight urokinase releases twofold more FPB than the low molecular weight species. In contrast, prourokinase does not release FPB until activated to urokinase. Contaminating thrombin or plasma is not responsible for urokinase-mediated FPB release because this activity is unaccompanied by FPA or B beta 1-42 cleavage, and is unaffected by heparin, hirudin, a monospecific antibody against thrombin, aprotinin, or alpha 2-antiplasmin. FPB release reflects a direct action of urokinase on fibrinogen because release is completely inhibited by a monospecific antibody against the enzyme. Further, urokinase releases FPB from the FPB-containing substrate B beta 1-42, thus confirming its specificity for the B beta 14 (Arg)-B beta 15 (Gly) bond. In addition to FPB release, SDS-PAGE analysis of the time course of urokinase-mediated fibrinogenolysis indicates progressive proteolysis of both the A alpha- and B beta-chains of fibrinogen that occurs after FPB release is completed. As a consequence of urokinase-mediated fibrinogenolysis, there is progressive prolongation of the thrombin clotting time. These studies indicate that urokinase has direct catalytic activity against fibrinogen. By releasing FPB, a potent chemoattractant, and by rendering fibrinogen less clottable by thrombin, urokinase may participate in processes extending beyond fibrinolysis.
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PMID:Urokinase has direct catalytic activity against fibrinogen and renders it less clottable by thrombin. 236 16

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.
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PMID:[Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates]. 240 Apr 5

Our previous study showed that an epitope defined by a monoclonal antibody against human urokinase is located on the 33-Kdalton catalytic domain of the enzyme (Nakamura, M. et al., Cell Struct Funct., 9, 167-179, 1984). The epitope structure was further determined and characterized on one-dimensional SDS-polyacrylamide slab gel maps of CNBr-cleaved polypeptide fragments as well as on their Western blots. A single homogeneous polypeptide with an approximate molecular weight of 3.4-Kdaltons was found to be antigenic. The monoclonal antibody exhibited a stronger inhibition of the enzyme activity than the polyclonal antibodies tested, and cross-reacted with a 65-Kdalton tissue-type plasminogen activator present in Detroit 562 cells. From these results and data made up with the help of a computer comparison of known sequences of urokinase and a tissue-type plasminogen activator, we concluded that the epitope is Cys-Gln-Gly-Asp-Ser-Gly-Gly-Pro-Leu-Val-Cys and contains a catalytically active residue, serine.
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PMID:A monoclonal antibody against human urokinase: the epitope structure and sequence homology with a human tissue-type plasminogen activator. 241 11

Rapid inhibition of urokinase in plasma obtained from women in the third trimester of pregnancy was assessed by the addition of 75 IU urokinase per ml plasma, and measurement of residual urokinase activity with PyroGlu-Gly-Arg-pNA after 5 minutes incubation at 37 degrees C. The urokinase inhibitory capacity was markedly increased for the pregnant women compared to non-pregnant controls. Alpha 1-antitrypsin, alpha 2-macroglobulin and alpha 2-antiplasmin did not account for the activity. Inhibition was higher for women with multiple gestations or macrosomia (n = 11) compared to normal pregnant women (n = 35) suggesting that the placenta contributes significantly to the measured activity. Inhibition was lower for women with hypertension (n = 33) compared to the normal pregnant women. Although the etiology for this difference is unclear, the decreased inhibitory activity may contribute to the increased risk for placental abruption that is observed for this group of women.
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PMID:The rapid inhibition of urokinase by plasma from pregnant women at risk for abruptio placenta. 243 78

Human urokinase (HUK) was purified from commercial product by high performance liquid chromatography on TSK GEL-G3000SW and affinity chromatography on benzamidine-Sepharose 4B. The purified enzyme was of a high molecular weight form (molecular weight of 53,000). This preparation was utilized as an antigen to immunize rabbits; the obtained antibody showed a high specificity against HUK. The antibody was conjugated to CNBr-activated paper disks. The antibody-conjugated paper disk and a fluorogenic peptide substrate, glutaryl-Gly-Arg-4-methyl-coumarine-7-amide, were used to measure urokinase (UK) activity in plasma. The calibration curve obtained by the proposed method passed through the origin and was linear in the range of 0-0.16 IU of HUK. The incubation of HUK with an excess amount of alpha 2-macroglobulin at 37 degrees C for 3 h gave only about a 30% decrease of the activity assayed by the proposed method. After incubations of HUK with alpha 1-antitrypsin and antithrombin III, the activity was completely inhibited. The incubation of HUK with plasma at 37 degrees C decreased the activity as a function of time. However, when the antibody-conjugated paper disk was used for the immunoreaction to HUK in plasma at 4 degrees C, no decrease of UK activity was observed. The plasma decay curve of UK activity after a single intravenous (i.v.) injection of HUK into a rabbit (12,000 IU/kg) indicated bi-exponential kinetics by using this assay method. The rate constants of the alpha and beta phases were 0.120 +/- 0.020 and 0.021 +/- 0.002 min-1, respectively. These result suggest that the proposed method is useful for measuring UK activity in plasma of patients with intravascular coagulation after i.v. administration of UK.
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PMID:Measurement of human urokinase activity in plasma by using mono-specific antibody-conjugated paper disk. 243 94

The protease inhibitor, aprotinin, has been examined for its ability to inhibit urokinase and tissue-type plasminogen activators at pH 7.4 in assays utilizing pyroGlu-Gly-Arg-p-nitroanilide and H-D-Ile-Pro-Arg-p-nitroanilide as substrates, respectively. Aprotinin inhibited both two-chain low molecular weight urokinase and the high molecular weight form of the enzyme in a competitive manner with a similar Ki (27 microM). There was no observable inhibition of tissue-type plasminogen activators at aprotinin concentrations up to 500 microM. These findings suggest that sensitivity to inhibition by aprotinin could be used to distinguish tissue-type and urokinase-type plasminogen activators.
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PMID:Aprotinin inhibits urokinase but not tissue-type plasminogen activator. 245 60

Hyperfibrinolytic states are reported to be a cause of bleeding in patients with amyloidosis. We reviewed the literature on excessive fibrinolysis in association with amyloidosis and report our findings from a patient with idiopathic amyloidosis who developed a bleeding diathesis. Coagulation laboratory studies indicated elevated plasminogen activator levels associated with a reduction of plasminogen and alpha 2-plasmin inhibitor (alpha 2-PI) levels. The level of tissue-type plasminogen activator (t-PA) inhibitor and t-PA antigen were normal. However, the patient did have a five- to sevenfold increase in amidolytic activity for the urokinase substrate pyro-Glu-Gly-Arg-pNA (S-2444). This case therefore represents a novel example of a hyperfibrinolytic state associated with amyloidosis caused by elevated urokinase-type plasminogen activator (u-PA). Epsilon-amino caproic acid (EACA) therapy resulted in an increase in alpha 2-PI and plasminogen levels and effectively reduced the blood loss. Hyperfibrinolytic states in amyloidosis have now been reported to be due to elevated t-PA and u-PA and depleted t-PA inhibitor.
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PMID:Elevated urokinase-type plasminogen activator level and bleeding in amyloidosis: case report and literature review. 249 17

A new assay method for measuring single-chain urokinase-type plasminogen activator (scu-PA) activity by quantitative calorimetric assay method is described. The serine enzyme u-PA activates plasminogen to plasmin, and scu-PA is a precursor protein obtained from a tissue culture of human kidney cells. The scu-PA solution was activated by plasmin to two-chain u-PA, which releases p-nitroaniline from pyro-Glu-Gly-Arg-pNA, and the optical density at 405 nm was measured. One unit of scu-PA was defined on the basis of 1 nmol of p-nitroaniline released from the substrate per second. One unit of scu-PA was found to be equivalent to 335 u-PA (two-chain) units (IU).
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PMID:Assay method for single-chain urokinase-type plasminogen activator. 253 34

Fresh human urine was found to contain at least three different molecular forms of fibrin-binding urokinase (UK) or its precursor, all of which were absorbed on a fibrin/Celite column at neutral pH, and could be eluted with 0.3-1.0 mol/l NaCl in phosphate buffer, followed by 0.2 mol/l, Arg, 2 mol/l KSCN, and 2 mol/l urea, respectively. The main molecular form isolated revealed a molecular weight (MW) of approximately 100,000 (UK-100), and the minor ones were estimated to have MW of 150,000-200,000 and 45,000. In contrast, commercially obtained UK preparations contained mostly active enzymes with MW of 53,000 and 32,000, respectively, and the remaining high molecular forms represented less than 2.0% of the total amount. Rabbit monospecific antibody (IgG) against UK subcomponent (active heavy chain; H-chain UK) reacted and inhibited the fibrinolytic activity of all the active UK molecules. The UK-100 isolated was relatively stable in solution at neutral pH and resistant to mild reduction, without molecular change. Although the preparation had a very low specific activity (ca. 300 IU/mg protein), both the pyro-Glu-Gly-Arg-pNA amidolytic and plasminogen activating activities could be partially enhanced by the addition of trace amounts of plasmin. In this process, the appearance of two additional active enzymes of MW 53,000 and 32,000 was also confirmed by zymography.
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PMID:Fibrin-binding urokinase (or precursor form of urokinase) with a molecular weight of about 100,000 in fresh human urine. 294 Dec 73


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