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)

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.
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PMID:The role of ovarian proteases and their inhibitors in ovulation. 255 99

Conditioned medium derived from the colon cancer cell lines was ineffective in solubilizing immobilized radiolabeled laminin. However, substantial degradation was observed in the presence of plasminogen and could be largely blocked by preincubation with polyclonal anti-urokinase antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized products generated either by the conditioned medium or by authentic urokinase supplemented with plasminogen yielded identical results. Analysis of the spent medium for urokinase by an enzyme-linked immunosorbent assay method revealed a similar profile to that achieved with the laminin degradation assays for the six cell lines tested. However, Northern analysis of urokinase-specific mRNA indicated that protein levels could not be entirely predicted by steady-state levels of the transcript. In a previous study, undifferentiated colon cancer cell lines expressed larger amounts of the plasminogen activator into the conditioned medium compared with their well-differentiated counterparts. However, these earlier studies were performed using cells grown in defined medium which lacked epidermal growth factor (EGF). EGF has been reported to affect plasminogen activator levels. Consequently, to investigate the role of EGF in the modulation of urokinase protein/activity, cell types representative of well- and poorly differentiated colon cancer were examined for their sensitivity of expression to this growth factor. In the absence of EGF, primitive cell types secreted, on average, 5 times more urokinase than their well-differentiated counterparts. In response to EGF, however, well-differentiated cell lines exhibited 4- to 6-fold increases in these parameters while the primitive cell lines were refractory to the peptide. Consequently, the differences in urokinase protein expressed by the well- and poorly differentiated groups of cells were abolished by the presence of EGF. The expression of a well-differentiated phenotype by colon cancer cell types in vivo probably depends to some extent on laminin within a basement membrane. The data presented herein are consistent with the idea that depletion of this glycoprotein from a basement membrane by urokinase-dependent mechanisms may contribute to the undifferentiated phenotype seen with many of these malignancies.
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PMID:Examination of urokinase protein/transcript levels and their relationship with laminin degradation in cultured colon carcinoma. 291 54

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.
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PMID:Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin. 294 36

Lp(a) represents a genetically transmitted class of plasma LDL having apo B-100 linked by a disulfide bridge to a glycoprotein, apo(a). Lp(a) is heterogeneous in size and density. Apo(a) is also heterogeneous in size (molecular weight between approximately 300,000 and 700,000) due probably to the polymorphism of both polypeptide and carbohydrate chains. Recent studies have shown that apo(a) has a striking amino acid sequence homology with plasminogen, a serine protease zymogen that following activation to plasmin enters the fibrinolytic system. Apo(a) is severalfold larger than plasminogen (molecular weight approximately 90,000) and also differs from it because it fails to be activated to plasmin. This is due to the fact that arginine is replaced by serine at the site of cleavage by streptokinase, urokinase, or tissue plasminogen activator. A single gene locus appears to control the Lp(a) polymorphism as well as the concentration of the Lp(a) phenotypes in the plasma. Patients with high plasma levels of Lp(a) have been shown to have an increased incidence of cardiovascular disease but a causal relationship has not been firmly established. The information that is being rapidly acquired on the structure of Lp(a) should facilitate the understanding of the molecular basis of the polymorphism of this genetic variant and of the role that the various Lp(a) phenotypes play in atherosclerosis and thrombosis. The potential physiologic role of Lp(a) remains open to inquiry.
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PMID:Lipoprotein(a): a genetically determined lipoprotein containing a glycoprotein of the plasminogen family. 297 66

The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products.
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PMID:Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells. 303 62

Urokinase-related proteins in human urine occur mainly as a 1:1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogenous glycoprotein of apparent Mr 50,000 was obtained with a yield of 40 micrograms/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 degrees C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with beta-mobility. Inhibition of urokinase occurred with a second order rate constant (k) of 8 X 10(3) M-1 s-1 in the absence and of 9 X 10(4) M-1 s-1 in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinase-type plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below 10(3) M-1 s-1 and thrombin with a k of 4 X 10(4) M-1 s-1 in the absence and 2 X 10(5) M-1 s-1 in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 +/- 0.7 micrograms/ml (mean +/- S.D., n = 25). The protein purified from plasma by immunoabsorption had the same Mr, amino acid composition, and immunoreactivity as the urinary protein. Furthermore, when urokinase was added to plasma, time-dependent urokinase-urokinase inhibitor complex formation was observed at a rate similar to that observed for the inhibition of urokinase by the purified inhibitor from urine. This urokinase inhibitor, purified from human urine, most probably represents a new plasma protease inhibitor.
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PMID:Purification and characterization of a novel inhibitor of urokinase from human urine. Quantitation and preliminary characterization in plasma. 309 4

The effects of plasmin have been examined because platelets may be exposed to plasmin in vivo and treatment of platelets with plasmin shortens platelet survival. Rabbit plasmin was prepared by urokinase activation of plasminogen immobilized on lysine-Sepharose. Plasmin caused rabbit platelets to aggregate and release the contents of their amine storage granules, but aggregation was slower than in response to ADP or thrombin. EDTA, prostaglandin E1, or creatine phosphate/creatine phosphokinase were inhibitory, but indomethacin was not. Deaggregation did not occur when platelets had been aggregated by a concentration of plasmin that caused extensive release of granule contents. EDTA or prostaglandin E1 caused deaggregation. Low concentrations of ADP and plasmin acted synergistically in causing platelet aggregation. Plasmin decreased the amounts of platelet membrane glycoproteins that stained with periodic acid-Schiff reagent; glycoprotein I was more susceptible than glycoprotein II and III. Concentrations of plasmin that induced the release of amine storage granule contents also released PAS-staining granule glycoproteins. Platelets incubated with plasmin, washed and resuspended, were not aggregated by ADP, but were aggregated strongly by the combination of fibrinogen and ADP, and bound 125I-fibrinogen to a greater extent than untreated platelets. Platelets preincubated with a high concentration of plasmin were unresponsive to thrombin, but were sometimes aggregated by fibrinogen. Plasmin decreased the buoyant density and increased the median size of platelets. Thus plasmin, as well as ADP and thrombin, may contribute to the density shift observed in platelets from rabbits in which thrombosis and continuous vessel injury have been induced.
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PMID:Effects of plasmin on rabbit platelets. 315 94

Although conventional biotechnology used for the synthesis of antibiotics, vitamins, amino acids, nucleotides, enzyme inhibitors and immunomodulating compounds has still a major impact in the production of pharmaceutical compounds, the importance of the new biotechnology is increasing. Whereas in conventional biotechnology naturally occurring strains are screened for production of pharmacologically active compounds, in new biotechnology known organisms are programmed by genetic engineering to produce a distinct protein or glycoprotein of human origin for substitution therapy. Such complex compounds from new biotechnology can be divided into products which might replace compounds which are already on the market by safer recombinant products such as human insulin, human growth hormone, urokinase, factor VIII and products which are new on the market such as interferons, lymphokines, tissue plasminogen activator, oligonucleotide probes, monoclonal antibodies and subunit vaccines. However, only a few of these recombinant products have reached the market such as human insulin, interferon alpha, interferon beta, human growth hormone and recombivax HB. In most cases, depending on the difficulties in demonstrating clinical efficacy, the investigated drugs have reached the marketing phase much faster than conventional chemical drugs. Return on investment of biotechnical produced pharmaceutics mainly depends on the issues of whether the product has to compete with chemically synthesized drugs, whether it is totally new but competes with other bioproducts, whether it is exceptional but the proof of clinical efficacy is difficult, or whether it is totally new and clinical studies are promising.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biobusiness in the pharmaceutical industry. 343 5

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.
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PMID:Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. 347 6

Human fibrosarcoma (HT-1080) cells, in contrast to normal fibroblasts, rapidly hydrolyze the glycoprotein, collagen, and elastin extracellular matrix (ECM) synthesized by cultured rat aortic smooth muscle cells. This degradation occurs at a rapid rate in the presence of serum, indicating that the cellular proteases responsible are relatively insensitive to serum proteinase inhibitors. Here it is shown that protease nexin I (PNI), a fibroblast-secreted inhibitor of urokinase, plasmin, and certain other serine proteinases, effectively inhibited the HT-1080 cell-mediated degradation of this ECM. PNI at 2.0 nM significantly inhibited matrix destruction for 1-2 days and at 0.2 microM caused a virtually complete inhibition that persisted for the entire 10-day period of observation. Inhibition of ECM destruction was accompanied by a transient arrest of HT-1080 cell proliferation that took place during the first 3 days after PNI addition. PNI did not inhibit the growth of normal fibroblasts and also did not inhibit the growth of HT-1080 cells that were seeded onto plastic dishes rather than onto ECM. Like many types of malignant cells, HT-1080 cells release large amounts of urokinase. Antibody against this plasminogen activator partially protected ECM from HT-1080 cell-mediated hydrolysis, indicating that it may have been a target of PNI. One potential physiological function of PNI could be to help maintain the integrity of connective tissue matrices, protection that malignant cells could overcome by secreting proteinases in excessive amounts.
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PMID:Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I. 351 69


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