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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Baby hamster kidney fibroblasts (BHK21 cells) transformed by polyoma or Rous sarcoma viruses aggregate less than the untransformed parental cells when incubated in growth medium in a gyratory shaker for 18-24 h. This difference can be measured by electronic particle counting, or by filtering aggregated suspensions of 32P-labelled cells through bolting fabric. The aggregation of transformed derivatives is not enhanced by the presence, during aggregation of epsilon-amino caproic acid, an inhibitor of plasmin activation. Some lines of transformed BHK21 cells do not appear less adhesive than untransformed cells in a short-term aggregation assay, and none adheres markedly less well when seeded onto homotypic cell sheets. The decreased aggregation of transformed cells is consistent with suggestions that LETS protein is involved in intercellular adhesion of fibroblasts as well as in attachment of cells to non-cellular substrates. If so, the short-term aggregation of freshly trypsinized cells may depend on secretion of LETS from an intracellular pool.
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PMID:On the reduced intercellular adhesiveness of virally transformed BHK21 cells. 15 24

The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium.
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PMID:Plasminogen-independent fibrinolysis by proteases produced by transformed chick embryo fibroblasts. 16 84

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

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.
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PMID:Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator. 22 74

We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA.
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PMID:Tumor promoters induce changes in the chick embryo fibroblast cytoskeleton. 57 62

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

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSVCEF) secrete a 70-kDa metallo-gelatinase at elevated levels over that of normal CEF. The 70-kDa enzyme has been purified from RSVCEF conditioned medium and represents 1-3% of the total protein in the RSVCEF conditioned medium. A 22-kDa protein, which appears to be the avian form of the tissue inhibitor of metalloproteases (TIMP), is co-isolated in association with the 70-kDa enzyme and can be separated from the enzyme by gel filtration carried out under denaturing conditions. The isolated 70-kDa species is in the zymogen form. It can be activated by treatment with the organomercurial, p-aminophenylmercuric acetate (APMA), yielding a 62-kDa active species derived by an apparent autoproteolytic cleavage from the 70-kDa proenzyme as determined by both substrate gel analysis and immunoblots using a monospecific antibody to the 70-kDa proenzyme. The proenzyme is poorly activated by trypsin and not activated by plasmin. The APMA-activated enzyme rapidly degrades denatured collagens but under identical conditions is unable to degrade native collagens, including basement membrane type IV collagen. Only at very high enzyme to substrate ratios (1:2) will native type IV collagen be hydrolyzed. Partial N-terminal amino acid sequencing of both the 70-kDa proenzyme and the 62-kDa active enzyme indicates that the avian enzyme is a member of the matrix metalloprotease family (MMP-2). When CEF cultures, infected with a temperature sensitive mutant of RSV, conditional for the expression of the transforming src oncogene, were incubated at the permissive and nonpermissive temperatures, differential levels of the 70-kDa enzyme were produced in direct proportion to the functioning of the src oncogene.
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PMID:Isolation and characterization of a 70-kDa metalloprotease (gelatinase) that is elevated in Rous sarcoma virus-transformed chicken embryo fibroblasts. 184 40

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

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.
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PMID:Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin. 282 45

The tumor induced RBC cytotoxicity assay has been used to explore the mechanism by which Rous sarcoma virus mutant transformed chick embryo fibroblasts and mouse 3T3 cells cause the cytolysis of RBC in vitro. All Rous sarcoma virus and viral mutant transformed cells were cytolytic for RBC. Three mutant viruses, tsGl251, rASV1702, and rASV157, appeared to cause quantitatively less cytolysis after transformation of chick embryo fibroblasts than other virally transformed cells. This decreased cytolytic activity may be correlated with decreased in vivo tumorigenicity. Temperature sensitive mutant transformed chick embryo fibroblasts and mouse 3T3, which were phenotypically normal and which secrete little if any plasminogen activator at non-permissive temperatures, were cytolytic at non-permissive temperatures. In addition, inhibitors of plasminogen activator and plasmin were ineffective inhibitors of cytotoxicity. The only effective inhibitor of cytotoxicity for both transformed chick embryo fibroblasts and mouse 3T3 cells was leupeptin. In Rous sarcoma virus transformed mouse 3T3 cells, the leupeptin inhibitable enzyme appears to be a plasma membrane enzyme.
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PMID:Role of proteases in red blood cell target cell destruction by cells transformed by Rous sarcoma virus mutants. 300 9


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