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:3.4.21.73 (urokinase-type plasminogen activator)
10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A direct rate assay for plasminogen activator has been developed using a synthetic fluorogenic peptide substrate, 7-(N-Cbz-glycylglycylargininamido)-4-methylcoumarin trifluoroacetate. The assay correlates well with the standard 125I-labeled fibrin plate assay using highly purified urokinase, culture fluids from WI-38, Chinese hamster vary or HeLa cells, or Rous sarcoma virus-transformed chick fibroblasts as the source of plasminogen activator. The assay is sensitive, rapid, and linear throughout a wide range of enzyme concentrations. With this substrate it is possible to determine inhibitor profiles for the various plasminogen activators, independently of the interfering potential of plasmin. All of the enzymes tested are inhibited by leupeptin and antipain but not by the related aldehydes, elastatinal and chymostatin. The macromolecular inhibitors soybean trypsin inhibitor and trasylol have little or no effect on the plasminogen activators tested. This substrate should be useful for the study of the effect of various agents on functional changes in cells secreting this enzyme and also should allow kinetic measurements of potential inhibitors.
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PMID:Direct fluorescent assay of urokinase and plasminogen activators of normal and malignant cells: kinetics and inhibitor profiles. 20 31

There is increasing evidence that urokinase secreted by tumor cells can be bound to a cell surface receptor retaining its full potential to activate plasminogen and subsequently cleave basement membrane constituents. This study was undertaken to discriminate between soluble and cell surface bound urokinase as a potential mediator of in vitro invasion by cultured colon cancer. Extracellular matrix invasion by a colon cancer cell line GEO, characterized as being a poor secretor of urokinase and having few receptors (less than 10(4) receptors/cell) was not augmented when these cells were made to secrete up to 8 times as much urokinase, in response to an exogenous urokinase gene driven by the Rous sarcoma virus long terminal repeat promoter. The majority of the plasminogen activator (greater than 95%) appeared in the culture medium, this reflecting the low numbers of binding sites displayed by GEO cells. In contrast, the cell line HCT 116 equipped with 10 times as many binding sites, (greater than 10(5)/cell), the majority of which are occupied with endogenous ligand, was an efficient invader of the extracellular matrix. Inhibition of urokinase binding to the cell surface receptors using an antibody to the A chain of the plasminogen activator reduced invasion by 65%. The cell line RKO is equipped with 3 x 10(5) receptors/cell, 15% of which are tagged with endogenous urokinase. Pretreatment of these cells with a concentration range of urokinase known to result in the majority of these binding sites being charged with the plasminogen activator led to a dose dependent increase in extracellular matrix invasion. Together, these data suggest that for cultured colon cancer, at least, invasion is a function of the amount of cell surface receptor bound urokinase.
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PMID:Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer. 164 43

Transformed cells produce elevated levels of the urokinase-type plasminogen activator (u-PA), which has been linked with the invasive or migratory phenotype of these cells. The u-PA is secreted and normally maintained in the inactive, single-chain form (scu-PA) and it has been assumed that natural activation occurs via a plasmin-mediated cleavage converting scu-PA to the active, two-chain form (tcu-PA). We now demonstrate that secreted scu-PA in Rous sarcoma virus-transformed chicken embryo fibroblast (RSVCEF) cultures is activated by an endogenous, plasmin-independent mechanism. Normal CEFs and CEFs infected with a temperature-sensitive RSV mutant and incubated at the nonpermissive temperature do not activate scu-PA. Conditioned medium harvested from plasmin-free cultures of RSVCEFs contains active tcu-PA as determined by two independent methods. The scu-PA is progressively converted with time in culture and requires the presence of intact cells or a plasma membrane-enriched fraction. When added to RSVCEF cultures, a synthetic peptide corresponding to residues 20-41 of the growth factor domain of chicken u-PA blocks the conversion to tcu-PA, and scu-PA accumulates in the cultures. These results suggest that scu-PA is secreted by cells, becomes bound to a u-PA receptor, and is proteolytically converted to active tcu-PA by a catalytic mechanism on the surface of RSV-transformed fibroblasts.
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PMID:Transformation-dependent activation of urokinase-type plasminogen activator by a plasmin-independent mechanism: involvement of cell surface membranes. 165 63

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.
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PMID:Serine protease and metallo protease cascade systems involved in pericellular proteolysis. 196 54

Secretion of urokinase-type plasminogen activator (uPA) by chicken embryo fibroblasts (CEF) is increased approximately 50-fold following transformation by Rous sarcoma virus (RSV). Using a cloned and fully sequenced chicken uPA cDNA probe, we have established that this increase in plasminogen activator production can be largely accounted for by an increase in cellular uPA mRNA. CEF contained on average less than 1 molecule of uPA mRNA/cell, whereas RSV-CEF contained 25-60 molecules/cell. The increase in cellular uPA mRNA levels was dependent on the activity of the RSV-encoded transforming protein, protein-tyrosine kinase pp60v-src. Cells infected with an RSV mutant encoding a temperature-sensitive form of the src protein (ts-NY68) contained low uPA mRNA levels when cultured at the nonpermissive temperature and high uPA mRNA levels when maintained at the permissive temperature. Temperature shift studies with tsNY68-CEF demonstrated that changes in pp60v-src activity rapidly altered uPA mRNA levels; the uPA mRNA content of total RNA extracts increased and decreased with half-time kinetics of 3-5 h. Serine/threonine-specific protein kinases also appear to modulate uPA mRNA levels in CEF cultures. Exposure of CEF and RSV-CEF for 24 h to the protein kinase C activating agent phorbol myristate acetate (PMA) increased cellular uPA mRNA levels to 20 and 260 molecules/cell, respectively. These data are consistent with the previously observed synergism between RSV and PMA in increasing plasminogen activator secretion. Nuclear run-on transcription analyses established that both RSV and PMA increase cellular uPA mRNA levels by way of increased uPA gene expression.
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PMID:Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. 215 28

We have prepared a conjugate of the plasminogen activator urokinase (UK) and ferritin, which maintains fibrinolytic activity. Monolayers of BALB/c-3T3 cells and of Rous sarcoma virus-transformed highly malignant line AA12-3T3, subcultured in plasminogen-free serum, were incubated with UK-ferritin at 0 degree and processed for transmission electron microscopy. Under these conditions, both of the lines showed specific receptors on the cell surface that were distributed in singlets, in small or large clusters. In the presence of excess native UK, the binding of ferritin was reduced by 99%, indicating the interaction of UK:ferritin with a specific receptor. The ligand-receptor interaction involves the catalytic site of UK, since the binding was completely impaired by preincubation of UK:ferritin with p-aminobenzamidine, a competitive inhibitor of the catalytic site of UK. The number and density of receptors decreased about one order of magnitude on the membrane of AA12 cells when compared with normal 3T3 cells. Saturation kinetics, using 125I-labeled UK, indicate the presence of 4 X 10(4) and 2.5 X 10(3) receptors on the membrane of 3T3 and AA12 cells, respectively. At 37 degrees, UK:ferritin redistributed on the plane of the membrane, in a process which was faster in malignant than in normal cells. Ferritin particles clustered in large groups on coated areas of the surface and were internalized by adsorptive pinocytosis. After 10 min at 37 degrees, the vesicles showed a progressively deeper internalization and a fusion with lysosomes, and some were observed in the Golgi complex area. Since the experiments were planned in order to exclude the presence of protease-nexin in the incubation medium, these data suggest the existence of a plasminogen-independent novel receptor for the catalytic site of plasminogen activators, the number on the cell surface of which decreases in Rous sarcoma virus-transformed mouse fibroblasts.
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PMID:Receptors for plasminogen activator, urokinase, in normal and Rous sarcoma virus-transformed mouse fibroblasts. 298 11

Previous studies have established that plasma cryoprecipitates of tumor patients, culture media of transformed cells and defined proteolytic fragments of fibronectin enhance the morphological cell transformation ( TEF activity) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We now report that purified human tissue type plasminogen activator (t-PA), but not urokinase (u-PA), has a similar TEF activity, at doses as low as 2 ng/ml (30 pM). Specific antibodies effectively neutralized the activity. No significant contamination (less than or equal to 1%) between the preparations of t-PA and fibronectin (FN) or its fragments ( FNdp ) was detected. The results suggest that t-PA may have a direct role in the process of morphological cell transformation in vitro.
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PMID:Tissue type plasminogen activator, but not urokinase, exerts transformation-enhancing activity. 653 4

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.
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PMID:A proenzyme from chicken plasma similar to human plasma prekallikrein. 655 13

The antifibrinolytic activity of cytosols obtained from cultured rabbit endothelial cells was studied to determine whether it resulted from the presence of an antiplasmin or an antiactivator. These cytosol preparations inhibited the fibrinolytic activity initiated by some plasminogen activators (e.g. urokinase, rabbit endothelial cell activator), but not others (e.g. activators associated with bovine endothelial cells and Rous sarcoma virus-infected chick embryo fibroblasts), suggesting that inhibition occurred at the level of plasmin formation, not plasmin activity. The fibrinolytic activity of plasmin itself was unaffected by concentrations of cytosol that completely blocked urokinase-mediated fibrinolysis consistent with this conclusion. In addition, the ability of urokinase to cleave 125I-plasminogen into its characteristic activation fragments was inhibited by cytosol in a dose-dependent manner. When urokinase was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two peaks of activity were detected, corresponding to Mr = 55,000 and 32,000. Urokinase preincubated with cytosol and analyzed in a similar manner demonstrated no activity in any portion of the gel, suggesting that its ability to function as a plasminogen activator was irreversibly lost following its interaction with cytosol. These results indicate that the antifibrinolytic activity of rabbit endothelial cells results from the presence of a molecule or molecules with antiactivator activity. The cellular location and unusual degree of specificity distinguish the endothelial cell inhibitor(s) from antifibrinolytic agents observed in other cells and in plasma and platelets.
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PMID:An inhibitor of plasminogen activator in rabbit endothelial cells. 678 54

Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF) constitute a well-characterized model system for oncogenic transformation, matrix degradation, and cancer invasion. As RSVCEF cultures employ both serine protease and metalloprotease cascades in the process of matrix degradation, they have contributed significantly to understanding the nature and regulation of these molecules involved in invasive cell behavior. RSVCEF produce elevated levels of a matrix metalloprotease-2 (MMP-2) whose hemopexin domain differs from mammalian MMP-2. The majority of MMP-2 produced by RSVCEF is present in a TIMP-free form which enhances its activation, catalytic activity and substrate specificity and therefore its matrix-degrading ability. RSVCEFs also exhibit high levels of urokinase-type plasminogen activator (uPA), which is found in active form in their conditioned medium in complete absence of plasminogen. Recombinantly expressed avian uPA is also in active form, while an active-site mutant of the same maintains its zymogen form, indicating the mechanism of activation of chicken uPA is autocatalytic. A domain and sequence comparison between chicken and human uPA attempts to identify motifs potentially responsible for the zymogen instability of avian uPA and its capability to autoactivate.
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PMID:What structure and function of avian plasminogen activator and matrix metalloproteinase-2 reveal about their counterpart mammalian enzymes, their regulation and their role in tumor invasion. 879 96


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