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)

Tissue and urokinase-type plasminogen activators are serine proteases with highly restricted specificity, their best characterised role being to release the broad specificity protease plasmin from inactive plasminogen. It has frequently been suggested that these, and similar proteases, are involved in axonal growth and tissue remodelling associated with neural development. To help define what this role might be, we have studied the expression of the plasminogen activators in developing rat nervous tissue. Urokinase-type plasminogen activator mRNA is strongly expressed by many classes of neurons in peripheral and central nervous system. We have analysed its appearance in spinal cord and sensory ganglia, and found the mRNA is detectable by in situ hybridisation very early in neuronal development (by embryonic day 12.5), at a stage compatible with it playing a role in axonal or dendritic growth. Tissue plasminogen activator mRNA, on the other hand, is expressed only by cells of the floor plate in the developing nervous system, from embryonic day 10.5 and thereafter. Immunohistochemical and enzymatic analysis showed that active tissue plasminogen activator is produced by, and retained within, the floor plate. A mechanism is suggested by which high levels of tissue plasminogen activator produced by the stationary cells of the floor plate could influence the direction of growth of commissural axons as they pass through this midline structure.
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PMID:The expression of tissue and urokinase-type plasminogen activators in neural development suggests different modes of proteolytic involvement in neuronal growth. 128 56

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin >> human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study. 129 2

The invasiveness of human gastric carcinoma cell lines (MKN45 and MKN28) in the subcutaneous tissue of nude mice and the degrading capacities of extracellular matrix (ECM) were studied. MKN45 cells were more invasive than were the MKN28 cells. Immunostaining revealed dense lamellar accumulation of fibronectin (FN) around the tumors. Along the front of the invasive MKN45 growth, however, the FN fibers were discontinuous and/or had completely vanished; the MKN28 tumor showed no FN fiber disconnection. ECM components other than FN never displayed such peritumoral massive accumulation. Cocultivation of human fibroblasts with MKN45 cells, more evidently than with MKN28 cells, revealed degradation of FN produced by fibroblasts in contact with each tumor. Both cell lines produced several FN-degrading enzymes in serum-free cultures. Proteases from the MKN45 medium were more active than were those of MKN28 in urokinase-type plasminogen activator (uPA) and metal-dependent serine proteinase-like proteases of 75 and 68 kDa in molecular weight (MW). Type I collagen-degrading 48-kDa protease was also detected from MKN45 medium but not from the MKN28 medium. MKN28 cells secreted other kinds of FN-degrading enzymes, estimated at approximate MWs of 29 and 100-150 kDa. We found no distinct differences in capacity to produce ECM components or ability to adhere to purified ECM components between these two cell lines. From these results we conclude that the stromal invasion of these cells into the subcutaneous tissue of nude mice is profoundly related to their FN-degrading capability. This capability may be catalyzed by uPA and/or metal-dependent serine proteinase-like proteases of 75 and 68 kDa.
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PMID:Fibronectin degradation by human gastric carcinoma cell lines and its associated proteases in relation to stromal invasion in nude mice. 129 40

Fluorescence-activated cell scanning analysis of human blood cells revealed novel urokinase receptors in large granular lymphocytes and a small subset of T-cells (CD3+). Culturing of T-cells with interleukin-2 to generate CD3+ lymphokine-activated killer cells caused a large increase in urokinase binding, suggesting that the urokinase receptor is an activation antigen. The receptor in lymphocytes was similar to that in monocytes with regard to size, affinity and ligand specificity, but did not mediate degradation of urokinase-inhibitor complexes. It is suggested that lymphocyte-bound pro-urokinase is activated, e.g. by the human T-cell-specific serine proteinase, HuTSP-1, and thereby starts a cascade of plasminogen activation important for extravasation of the cells.
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PMID:Identification and characterization of urokinase receptors in natural killer cells and T-cell-derived lymphokine activated killer cells. 131 84

Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family.
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PMID:Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour. 131 62

The potential contribution of serine/threonine-specific protein phosphatases in the transcriptional regulation of plasminogen activator and plasminogen activator inhibitor gene expression was explored in human HT-1080 fibrosarcoma and U-937 monocyte-like cells using okadaic acid, a potent and specific inhibitor of phosphatases 1 and 2A (PP1 and PP2A). In both cell types okadaic acid induced plasminogen activator type 2 (PAI-2) gene transcription and mRNA and potentiated induction mediated by phorbol-12-myristate-13-acetate and tumor necrosis factor. Okadaic acid-mediated induction of PAI-2 was inhibited by 8-bromo-cAMP in HT-1080 cells but not in U-937 cells. Okadaic acid had opposite effects on urokinase (u-PA) gene expression in the two cell lines; u-PA mRNA and gene transcription was suppressed in HT-1080 cells but transiently induced in U-937 cells. Tissue-type PA (t-PA) mRNA, although undetectable in U-937 cells, was also suppressed by okadaic acid in HT-1080 cells. This effect was selective, as constitutive and phorbol-12-myristate-13-acetate-mediated expression of plasminogen activator inhibitor type 1 mRNA was not modulated by okadaic acid in either cell type. These results indicate that PP1 and PP2A protein phosphatases are involved in signal transduction pathways modulating PAI-2, u-PA, and t-PA, and furthermore, that okadaic acid interaction with the protein kinase C and A pathways are gene- and cell type-specific.
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PMID:Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. Studies on the plasminogen activating system. 131 13

The binding of urokinase-type plasminogen activator (uPA) to its specific cell-surface receptor (uPAR) localises the proteolytic cascade initiated by uPA to the pericellular environment. Inhibition of uPA activity or prevention of uPA binding to uPAR might have a beneficial effect on disease states wherein this activity is deregulated, e.g. cancer and some inflammatory diseases. To this end, a bifunctional hybrid molecule consisting of the uPAR-binding growth-factor domain of uPA (amino acids 1-47; GFuPA) at the N-terminus of plasminogen-activator inhibitor type 2 (PAI-2) was produced in Saccharomyces cerevisiae. The purified protein inhibited uPA with kinetics similar to placental or recombinant PAI-2 and was also found to bind to U937 cells and to FL amnion cells. GFuPA-PAI-2 competed with uPA, the N-terminal fragment of uPA and a proteolytic fragment of uPA (amino acids 4-43) in cell binding experiments, indicating that the molecule bound to the cells via uPAR. Hence, both the uPA-inhibitory and uPAR-binding domains of the hybrid molecule were functional, demonstrating the feasibility of the novel concept of introducing an unrelated, functional domain onto a member of the serine-protease-inhibitor superfamily.
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PMID:A hybrid protein of urokinase growth-factor domain and plasminogen-activator inhibitor type 2 inhibits urokinase activity and binds to the urokinase receptor. 132 Oct 39

An enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 +/- 0.6 ng/ml (mean +/- SD, n = 27). The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising AA 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with alpha 2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA. 137 17

Effects of water-miscible organic solvents added to an aqueous buffer on the activity of several serine proteinases were studied. Plasmin in particular showed a dramatic difference in activity depending on the hydrophobicity of the added organic solvent through a combination of Km and kcat effects. An inverse linear correlation between the polarity of the mixed solvent and the log (kcat/Km) of plasmin activity was observed for both H-D-Val-Leu-Lys-p-nitroanilide (S-2251) and pro-urokinase as the substrate. The activity of plasmin was less dependent on the polarity of the added solvent when other chromogenic substrates were employed that contained an arginyl residue in the P1 site.
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PMID:Medium effects on the kinetics of human plasmin. 138 9

Proteinase species secreted by 10 human gastric carcinoma cell lines were analyzed by gelatin zymography and immunoblotting. These cell lines were classified into the following three groups with respect to proteinase secretion: cell lines secreting mainly gelatinases A and/or B; those secreting multiple types of serine proteinases; and those scarcely secreting these enzymes. Two cell lines of the second group, STKM-1 and MKN28, hardly secreted metalloproteinases but secreted the following four types of serine proteinases: (a) two trypsin-like enzymes (M(r) 26,000 and 24,000 in proenzyme forms); (b) a tissue kallikrein-like enzyme (M(r) 150,000 in a complex form); (c) a plasmin-like enzyme (M(r) 70,000); and (d) a plasminogen activator (urokinase-type, M(r) 57,000, from STKM-1 and tissue-type, M(r) 70,000, from MKN28). The M(r) 70,000 plasmin-like enzyme was also detected at lower levels in the conditioned media of four other cell lines (MKN1, MKN45, NUGC-3, and KATO III). The M(r) 24,000 proenzyme of the trypsin-like enzyme was purified from the serum-free conditioned medium of STKM-1. The proenzyme was activated by enterokinase treatment or autolytically by incubation at neutral pH, decreasing its apparent molecular weight from 24,000 to 23,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activated enzyme extensively degraded fibronectin, laminin, and gelatins and to lesser extents type I, III, IV, and V collagens at 30 degrees C. These results suggest that the matrix serine proteinases may play a major role in the matrix degradation by some kinds of human cancer cells.
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PMID:Multiple secretion of matrix serine proteinases by human gastric carcinoma cell lines. 138 87


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