Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue-type plasminogen activator (t-PA) is a serine protease with a molecular weight of about 70,000. It activates plasminogen to plasmin by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis showed that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate. The directed action of plasmin towards fibrin in vivo can be explained by the low Michaelis constant in the presence of fibrin (0.16 microM) which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface is protected from rapid inactivation by alpha 2-antiplasmin. Studies on the thrombolytic properties of t-PA (purified from melanoma cell cultures or obtained by recombinant DNA technology) in various animal models and in selected patients revealed that t-PA is a specific thrombolytic agent which induces thrombolysis without causing extensive systemic activation of the fibrinolytic system.
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PMID:Tissue-type plasminogen activator. 295 11

We have carried out enzymatic, immunofluorescence, and surface iodination studies which show that B16 melanoma cells express the single chain form of the urokinase type plasminogen activator (uPA) on their cell surface, and that these cells are capable of plasminogen-dependent fibronectin degradation. The significance of the expression of surface single-chain uPA and uPA activity to the metastatic process was examined by preincubating melanoma cells with uPA modulating agents followed by i.v. injection of the cells into mice and enumeration of pulmonary nodules 17 days later. B16 cells that had been pretreated with anti-uPA immunoglobulins that were inhibitory to uPA activity invariably showed significantly decreased numbers of metastases compared to controls. On the contrary, pretreatment with plasmin, which is not only the product of the uPA catalyzed reaction but is also able to convert single-chain uPA to uPA, significantly increased the numbers of metastases. Control treatments, which included normal rabbit and mouse immunoglobulins, monovalent noninhibitory anti-uPA Fab fragments, and various monoclonal and polyclonal antibodies directed against other B16 cell surface antigens, did not affect the metastatic potential of the cells. Divalent inhibitory anti-uPA F(ab)2 fragments, on the contrary, inhibited metastasis as efficiently as intact IgG. The results support the hypothesis that proteolysis of extracellular matrix components by cell surface-localized uPA may be a critical step during the process of tumor cell invasion and metastasis.
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PMID:Modulation of metastatic potential by cell surface urokinase of murine melanoma cells. 296 89

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.
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PMID:Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment. 296 24

The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.
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PMID:Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline. 299 28

We have studied the regulation by glucocorticoids and dibutyryl cAMP of the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and a Mr approximately 54000 plasminogen activator inhibitor accumulated in serum-free conditioned culture fluid by a human fibrosarcoma, a human glioblastoma and a human melanoma cell line (HT-1080, UCT/gl-1 and Bowes). For the quantitation of u-PA and t-PA, we used sandwich-type ELISA with a combination of polyclonal and monoclonal antibodies. For an estimation of variations in the amount of the inhibitor, we used sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by Coomassie blue staining of conditioned culture fluid proteins, the inhibitor protein band being identified by its selective removal by passage of the conditioned culture fluids through a column with monoclonal antibodies against the inhibitor. The modulation of the 3 proteins by the hormonal agents varied greatly between the cell lines. The proteins were independently regulated, in the sense that the hormonal agents did not concomitantly change their levels in the direction expected either to increase or decrease total extracellular plasminogen activator activity. In conditioned culture fluids containing both t-PA and inhibitor, the two were present in the medium as a Mr approximately 120 000 complex. In contrast, no u-PA inhibitor complexes were found in conditioned culture fluid from any of the cell lines; this is likely to be due to the occurrence of u-PA in the culture fluid in the one-chain proenzyme form, which, unlike active u-PA, does not react with the inhibitor. These findings illustrate the complexity of the regulation of extracellular plasminogen activator activity, and imply that the presumed functional diversity of u-PA and t-PA may be related to their independent regulation.
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PMID:Hormonal regulation of extracellular plasminogen activators and Mr approximately 54,000 plasminogen activator inhibitor in human neoplastic cell lines, studied with monoclonal antibodies. 301 58

We have used two kinds of vectors to express a cDNA of human tissue plasminogen activator (t-PA) in mammalian cells. In one case, cDNAs inserted into vectors based on bovine papilloma virus were introduced into cultured murine cells and cell lines were established that efficiently and continuously secrete enzymatically active t-PA into the medium. Second, the t-PA gene was used to replace the sequences of the simian virus (SV40) genome that code for the viral coat proteins. Virus stocks were generated and used to infect a stable line of cultured simian cells. During the resulting lytic infection, expression of the t-PA gene is governed by the potent SV40 late promoter and enzymatically active t-PA accumulates rapidly in the medium. We have used these two vector systems to analyze the biosynthesis and transport of recombinant t-PA and to compare its properties with those of "natural" t-PA secreted by the Bowes line of human melanoma cells. t-PA secreted from all three sources is identical in specific activity (approximately 20,000 units/mg) despite differences in patterns of terminal glycosylation. Furthermore, non-glycosylated t-PA synthesized in the presence of tunicamycin was secreted efficiently and was indistinguishable in specific activity from glycosylated t-PAs.
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PMID:Expression of human tissue-type plasminogen activator from lytic viral vectors and in established cell lines. 303 88

An inhibitor of plasminogen activator (PA) secreted by a tumorigenic, but non-metastatic, rat mammary adenocarcinoma cell line has been purified to apparent homogeneity and characterized. It strongly inhibited human urokinase, but was 100 times less potent in inhibiting bovine trypsin and had no effect on plasmin or thrombin. A secreted, urokinase-type PA (Mr 48 000) and a cell-associated PA from a metastatic rat adenocarcinoma cell line were also strongly inhibited. In contrast, a tissue-type PA (Mr 66 000), secreted by human melanoma cells, was only slightly inhibited. Purified inhibitor showed a band of Mr 66 000 in sodium dodecyl sulphate/polyacrylamide gel electrophoresis and an isoelectric point of 4.5 after chromatofocusing. The inhibition of human urokinase was non-competitive.
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PMID:Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells. 308 43

When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.
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PMID:Influence of carbohydrate side chains on activity of tissue-type plasminogen activator. 308 8

Polyclonal and three monoclonal anti-tissue plasminogen activator (a-t-PA) antibodies (1:3 C5, 1:3 G5 and 2:2 B10) were characterized by using enzyme immunoassay (EIA) in which beta-D-galactosidase was coupled to a-t-PA antibody. Monoclonal antibodies called 2:2 B10 and 1:3 G5, specific for both one-chain and two-chain t-PA, strongly bound to one-chain t-PA purified from cultured melanoma cell lines, but 1:3 C5 antibody bound weakly to such t-PA. When polyclonal t-PA antibody was used as the first reaction antibody immobilized on silicone pieces, few determinants were available for monoclonal antibody used in the second reaction due to previous interaction of these determinants in the first reaction. When t-PA levels in the plasma were determined, the presence of EDTA enhanced the sensitivity of t-PA determination by the present EIA technique. Plasma concentrations of t-PA were measured to be higher with 2:2 B10 monoclonal antibody than with polyclonal antibody as the first antibody. Tissue-PA was mainly detected in the endothelial cells, but not in the muscular layer of the inferior mesenteric artery when immunochemical technique was used with polyclonal t-PA antibody.
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PMID:Characterization of various antibodies against tissue plasminogen activator using highly sensitive enzyme immunoassay. 308 76

Messenger RNA of the phorbol ester-induced 48kDa protein from human melanoma cells (Bowes) was isolated, characterized and used to study the protein processing. The 48kDa mRNA is induced simultaneously with that of tissue-type plasminogen activator. This induction is prominent as shown by sedimentation profiles on linear sucrose gradients. The mRNA can be isolated by classical phenol extractions, has a poly(A)-tail and sediments with a coefficient of 20 S. Translation in reticulocyte lysates yields a 48kDa protein whether the translation is modified with canine pancreas microsomal membranes or not. Analysis of 48kDa mRNA translation products by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that the phorbol ester-induced 48kDa is a monomeric one-chain polypeptide. Glycosylation could not be detected, nor signal peptide cleaving, suggesting that it is a non-secreted intracellular protein.
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PMID:Messenger RNA for a phorbol-ester induced 48,000 dalton protein from human melanoma cells. 308 63


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