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)

A monoclonal antibody has been raised against the serine protease, plasminogen activator (PA) produced by Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF), and selected for its ability to inhibit the catalytic activity of PA. The high specificity and anticatalytic nature of the antibody has allowed probing of the direct role of PA in cellular behavior. Microgram quantities of monoclonal IgG inhibit the overgrowth and the morphological changes associated with RSVCEF transformation and the degradation of extracellular matrix mediated by RSVCEF, indicating a catalytic role for PA in these cellular processes. Specific cleavage of extracellular matrix proteins by immunoaffinity-purified PA in the complete absence of plasminogen demonstrates a direct catalytic involvement of PA in matrix degradation.
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PMID:An anticatalytic monoclonal antibody to avian plasminogen activator: its effect on behavior of RSV-transformed chick fibroblasts. 301 Dec 82

Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.
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PMID:Proliferation of a human epidermal tumor cell line stimulated by urokinase. 303 46

Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.
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PMID:Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes. 309 95

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.
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PMID:Structure and function of human tissue-type plasminogen activator (t-PA). 309 31

Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275----Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-p-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275----Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with alpha 2-macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has an increased fibrin binding compared to the two-chain form.
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PMID:Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis. 310 80

We have determined that the primary reason for the frequently encountered poor survival of human scirrhous breast carcinomas in short-term (4 days) organ culture is mechanical injury to the tumor tissue during explant preparation. It was possible to minimize this injury by preparing 0.5-mm-thick slices using very sharp blades. This resulted in much improved preservation of tissue structure and function, as assessed by histology, DNA content, and enzyme synthesis and secretion. With the exception of insulin, which was always present in the culture medium, exogenous hormones, including estrogen, or serum did not further improve explant preservation. In rodent mammary tumors, growth in vivo and production of the serine protease plasminogen activator (PA) in organ culture are coordinately regulated by hormones, suggesting that PA may be a valuable indicator of tumor hormone responsiveness. We have now tested the effect of estrogen and other hormones on PA secretion in organ cultures of primary human breast carcinomas. We found that: modulation of PA by 17-beta-estradiol (10-8) M) occurred only in carcinomas which were positive for both estrogen and progesterone receptors; of 21 such tumors, 11 (52%) were responsive. Plasminogen activator was not modulated by estradiol in any of the 22 tumors which were negative for one or both receptors; hydrocortisone (10(-7) M) effectively inhibited, and 3,5,3'-L-triiodothyronine (10(-8) M) and adenylate cyclase activators effectively stimulated PA in most breast tumors, regardless of their estrogen and progesterone receptor status. Prolactin (5 micrograms/ml) had no effect when tested alone; urokinase-type PA was found to be the principal PA produced by human breast tumors. Changes in its rate of synthesis and secretion and not in the content of PA inhibitors appeared to be the prevailing mechanism of enzyme regulation by hormones. In summary, short-term organ culture coupled with the use of PA as an index of response appears to be a promising approach to the study of hormone sensitivity of primary human breast carcinomas.
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PMID:Hormonal modulation of plasminogen activator: an approach to prediction of human breast tumor responsiveness. 310 11

The activity of the serine protease plasminogen activator (PA), which correlates with tumorigenicity and metastatic capacity, was examined using the 125I-labeled fibrin plate assay in cell extracts from four human glioma lines as a function of growth in vitro. Cell-associated inhibitory activity to plasmin and urokinase-type PA was also measured concurrently. The relative PA activities differed markedly among the lines, whereas inhibitory activities did not. Two lines, SNB-19 and SNB-75, exhibited maximal PA activities (1-6 m Plough units/micrograms protein) as cultures approached confluence, whereas two other lines, SNB-56 and SNB-78, expressed low PA activity at all times (less than 0.2 m Plough units/micrograms protein). The PA of SNB-19 cell extracts was predominantly urokinase-type PA. In addition to having the highest PA levels, SNB-19 and SNB-75 were the most clonogenic in soft agar and tumorigenic in nude mice. In contrast, SNB-56 and SNB-78 were poorly clonogenic in soft agar and were not tumorigenic in nude mice. Measured directly, inhibitory activities to plasmin, urokinase-type PA, and tissue-type PA were detected in SNB-19 (high PA) and SNB-56 (low PA) cell extracts. However, there were no qualitative or quantitative differences in inhibitor effects between SNB-19 and SNB-56 suggesting that the differences in PA activity between these lines resulted from changes in PA activity and were not due to differential plasminogen activator inhibitor effects. The ability of the differentiating agent sodium butyrate (NaB) to modulate total PA activity was also examined. Peak SNB-19 cell PA activity was decreased in a concentration (Ki, 0.75 mM) and time-dependent manner by the addition of nontoxic amounts of NaB. The dose-dependent decrease in PA activity induced by NaB was most likely due to an effect on PA itself, since the action of inhibitor on urokinase was unchanged in response to NaB. These results suggest that net cellular PA activity in glioma cells is a balance between relative PA activity and inhibitor(s) effects and that this balance can be modulated by sodium butyrate.
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PMID:Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. 312 Nov 70

Tissue-type plasminogen activator (t-pa) is a serine protease comprising four different putative structural domains with homologies to fibronectin finger-like structures (finger), epidermal growth factor, kringle structures, and the active site of serine proteases. Only the finger and epidermal growth factor domain are each entirely encoded by unique single exons. We assessed the functional contribution of these two structural domains by making mutants precisely deleted for one or both of the relevant exons. The three mutant genes were expressed in monkey cells, and the variant proteins, purified from the culture medium, were characterized for their fibrinolytic activity, fibrinogenolytic potential, and affinity for fibrin. No significant difference in any biochemical property was observed among the variants. All three variants retained a catalytic dependence on cyanogen bromide fragments of fibrinogen which could not be distinguished from the wild-type enzyme. The activities of the variants were also very similar to that of wild-type t-pa, showing no detectable fibrinogenolytic potential in human plasma at activator concentrations of 500 IU/ml, or when their fibrinolytic activity was tested in human plasma using the 125I-labeled fibrin clot lysis assay at activator concentrations of 150 IU/ml or greater. However, the variants were markedly defective in fibrinolysis at low activator concentrations such that essentially no fibrinolysis was detected at 15 IU/ml. Measurement of fibrin binding showed that the variants lacked the high fibrin binding characteristic of wild-type t-pa. These results demonstrate that the fibrin specificity and fibrin-dependent activity of t-pa are independent of the protein's high affinity for fibrin. The implication of these results is that the t-pa variants would be ineffective activators at a physiological concentration of approximately 2 IU/ml but would be expected to behave similarly to wild-type t-pa at the steady-state plasma concentrations of 0.75-1.25 micrograms/ml (approximately 500 IU/ml) currently required for coronary reperfusion in patients receiving t-pa for acute myocardial infarction (Garabedian, H.D., Gold, H.K., Leinbach, R.C., Yasuda, T., Johns, J.A., and Collen, D. (1986) Am. J. Cardiol. 58, 673-679).
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PMID:Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin finger-like and/or the epidermal growth factor domains. 312 18

The serine protease tissue-type plasminogen activator (t-PA) is synthesized by murine oocytes undergoing meiotic maturation, but not by arrested primary oocytes. Dormant, stable t-PA mRNA accumulates during oocyte growth, so that fully grown, arrested primary oocytes contain in their cytoplasm approximately 10,000 copies of this molecule. Translation of t-PA mRNA is triggered upon resumption of meiosis and is accompanied by a progressive and concerted increase in its size. This structural change can be accounted for by increased polyadenylation at the 3' end of the molecule. Following its translation, t-PA mRNA is degraded; it is no longer detectable in fertilized eggs. The identification of a dormant mRNA in murine oocytes and the demonstration that its translational activation is accompanied by elongation of its poly(A) tail may provide insights into the control of gene expression during meiotic maturation and early mammalian development.
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PMID:Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA. 312 15

Tissue-type plasminogen activator (t-PA) is a mosaic protein containing several distinct structural domains attached to the serine protease catalytic unit present at its COOH terminus. To investigate structure-function relationships in t-PA, we deleted the NH2-terminal domains, finger and epidermal growth factor, by genetic engineering. The genes for the parent and mutant t-PA were expressed in a bovine papilloma virus-dependent mammalian cell system. The secreted proteins were purified to homogeneity. The mutant protein was processed to the expected size of about 60 kDa compared to approximately 68 kDa for the parent t-PA, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography. While the mutant t-PA had amidolytic activity comparable to native t-PA, it did not bind appreciably to fibrin. Consequently, fibrin-dependent enzymic activity, i.e. plasminogen activation in the presence of soluble fibrin and fibrinolysis were lower than with native recombinant t-PA. The effect of deletion of NH2-terminal domains on the plasma half-life (t1/2) was investigated by injecting native and mutant t-PA into mice. While the majority of the t-PA disappeared initially with a t1/2 of about 2 min, mutant t-PA cleared at a much slower rate with t1/2 of about 50 min. These findings suggest that the NH2-terminal domains of t-PA not only determine its specificity for binding to fibrin but also mediate its clearance from plasma in vivo. Furthermore, the catalytic unit in t-PA seems to function autonomously.
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PMID:Structure-function analysis with tissue-type plasminogen activator. Effect of deletion of NH2-terminal domains on its biochemical and biological properties. 312 83


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