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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The results of four different assay methods showed that both normal and malignant plasminogen activator-secreting cells deposited substantial amounts of this protease on tissue-culture substrata, including collagen coatings. The cells studied were Rous sarcoma virus (RSV)-transformed vole fibroblasts, a malignant neural cell line (NG108-15) capable of neurite formation, and normal mouse-regenerating sensory neurons. Deposited plasminogen activator was detected by a fibrin overlay assay at sites from which cells growing on coverslips had been gently dislodged, showing that active enzyme is left beneath cells and in the immediate pericellular area. For neuronal cells, fibrinolytic zones were detected not only at the previous positions of cell bodies but also along the terrain conditioned by neurite extension, suggesting that a trail of plasminogen activator is left behind during growth cone movement. Substratum-bound enzyme could be solubilized in buffers containing sodium dodecyl sulfate (SDS) or Triton X-100 and demonstrated by zymography following electrophoresis or assayed for amidolytic activity with a chromogenic substrate (Kabi S-2251). The results suggest that plasminogen activator may be considered a component of substrate-adhesion material. Secretory proteases deposited directly on matrix molecules would seem strategically positioned to participate in local degradation of components of the extracellular environment.
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PMID:Normal and malignant cells, including neurons, deposit plasminogen activator on the growth substrata. 352 28

A direct assay for plasminogen activator (PA) was developed. It employed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and beta-mercaptoethanol to monitor PA-mediated conversion of single chain, 125I-plasminogen to two chain plasmin. By incorporating Triton X-100, albumin and trasylol in the reaction buffer, we were able to minimize the adsorptive and autolytic loss of reactants frequently associated with similar approaches. Under these conditions, plasmin formation was linear for at least 24 hours, dose-dependent over a 20-fold range of urokinase concentrations, and at least 100-fold more sensitive (0.05 units/ml) than previously reported direct assays for PA. The versatility of the assay was demonstrated by its ability to distinguish between urokinase-like and tissue-type PA, and to quantitate the effects of agents like fibrin and epsilon-amino caproic acid on their respective activities. The assay was readily adapted to detect inhibitors of PA in various samples, and was employed to demonstrate the presence of such inhibitors in both rabbit and bovine endothelial cells. Interestingly, the rabbit inhibitor was found to block the activity of urokinase but not that of tissue-type PA, while the bovine inhibitor neutralized the activities of both molecules. These results demonstrate that cleavage of 125I-plasminogen can be employed as a direct, sensitive and quantitative assay for various PAs, and thus offers a new approach for studying plasminogen activation and agents that stimulate or inhibit it.
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PMID:A direct, plasmin-independent assay for plasminogen activator. 623 50

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced "down-regulation" of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation.
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PMID:Down-regulation of epidermal growth factor receptor correlates with plasminogen activator activity in human A431 epidermoid carcinoma cells. 630 Sep 5

Metastatic malignant melanomas from 16 patients, extracted with Triton X-100, were analyzed for plasminogen activator activity by azocaseinolysis . In 6 cases tumor explants were set up also in short-term organ culture, and the rate of plasminogen activator secretion into the culture medium was determined. Both the extractable activator content [8.66 +/- 7.8 "Committee on Thrombolytic Agents" (CTA) U/g tissue] and the activator secretion rates (0.90 +/- 1.6 CTA U/g/hr) were low in comparison with values for other human tumors. In addition to the activity, the type of plasminogen activator also was determined by immunoinhibition with goat antihuman urokinase antibody in the azocaseinolytic assay, as well as by sodium dodecyl sulfate (SDS) gel electrophoresis followed by zymography on fibrin-agar, in the presence and absence of antibody. On the average, 77% of the activator activity was of the urokinase type in the extracts, and 90% in the culture fluids. Immunoperoxidase reaction for the detection of urokinase showed this enzyme to be localized mainly in the cell membrane of the melanoma cells; stromal elements showed no specific staining. These results are of interest in view of the findings made recently by investigators in several laboratories that in all but one of the melanoma cell cultures derived from metastatic human tumors, only the vascular type ("tissue activator") was cell associated or was secreted into the culture medium. The possible reasons for this discrepancy are discussed.
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PMID:Plasminogen activators in human malignant melanoma. 637 38

A cytokine that inhibits fibrinolysis has been detected in the serum-free culture medium of guinea pig and hamster fibroblasts. This proteinase inhibitor was also present in Triton X-100 extracts of guinea pig cells. It was stable at pH 3.0 for 2 hr and was produced by cells rather than assimilated from serum in the culture medium as evidenced by: (a) an apparent molecular size (less than 45 kilodaltons) less than that of the principal serum-derived proteinase inhibitors; (b) its continued secretion after several passages of the cells in serum-free medium; and (c) the lack of inhibitory activity in the medium of mitomycin C-treated cells. The cytokine inhibited the proteinase activity of human urokinase, soluble TPA-stimulated guinea pig plasminogen activator, and the cell-associated plasminogen activator of tumorigenic guinea pig cells. Soluble plasminogen activator appeared to be inhibited to a greater degree than the cell-associated enzyme. The fluorogenic substrate (7-(N-carbobenzoxyglycylglycylargininamido)-4-methylcoumarin was used in a direct assay of proteinase activity and demonstrated that the cytokine inhibited both plasminogen activator and plasmin, the two proteinases of the fibrinolytic cascade. Tumorigenic guinea pig and hamster fibroblasts as well as nontransformed guinea pig fibroblasts were found to produce the inhibitory cytokine, and the amount of inhibitor secreted was independent of the tumorigenic potential of the cells. Production of the inhibitor by normal cells may be related to contact inhibition of growth, and this cytokine may contribute to the fine regulation of local proteolysis within tissues.
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PMID:Secretion of proteinase inhibitors by tumorigenic and nontumorigenic guinea pig and Syrian hamster fibroblasts: evidence for autocrine regulation of local proteolysis. 642 73

A urokinase-type plasminogen activator secreted by subcultured normal human umbilical vein endothelial cells was purified and compared to urinary urokinase (Mr = 54,000). The enzyme was isolated from serum-free conditioned medium in the presence of 0.1% (v/v) Triton X-100 by p-aminobenzamidine-agarose affinity chromatography, followed by Sephacryl S-200 gel filtration, followed by immunoadsorption chromatography on affinity purified specific anti-urokinase IgG-Sepharose CL-4B. This plasminogen activator form was obtained from the culture medium with a yield of about 47% and specific activity of about 93,000 IU/mg of protein, and represented approximately 18% of the total multiple molecular plasminogen activator activity forms present in endothelial cell conditioned medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single band of plasminogen activator activity with an estimated molecular weight of about 54,000 that was completely inhibited by diisopropyl fluorophosphate (DFP) as well as a single band of radioactivity with similar molecular weight for both the isolated L-[4,5-3H]leucine and [3H]DFP-labeled enzyme. The radiolabeled protein focused as a single major band with a pI value of pH 8.5. The endothelial cell activator and urokinase appeared to be identical in terms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, location of the [3H]DFP-labeled active site in the Mr = 33,000 heavy chain and [3H]DFP-labeled active site tryptic peptide, and two-dimensional 125I-labeled tryptic peptide maps. In quenching experiments of the fibrinolytic activities using affinity purified specific anti-urokinase IgG the endothelial cell-derived activator and urokinase appeared to be immunochemically identical, but unrelated to tissue plasminogen activator. These results indicate that the Mr = 54,000 urokinase-type plasminogen activator from cultured normal human endothelial cells is similar to, or identical with, Mr = 54,000 urinary urokinase.
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PMID:Isolation and characterization of a urokinase-type plasminogen activator (Mr = 54,000) from cultured human endothelial cells indistinguishable from urinary urokinase. 653 33

Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.
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PMID:Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. 657 65

In inflammatory macrophages, plasminogen activator exists in two active forms, a soluble form released into the extracellular medium and a cell-associated form. This communication describes some properties of the cellular form of plasminogen activator, in intact macrophages and in cell lysates. Cellular plasminogen activator is a membrane protein, associated with the outer face of the plasma membrane; in intact macrophages, it participates in the activation of exogenous plasminogen and, thus, has to be considered as an ectoenzyme. A plasminogen activator activity can be detected in cell lysates (macrophage monolayers lysed in 0.1% Triton X-100) only when plasmin production is followed by the use of small synthetic substrates because a soluble inhibitor, released during extraction, blocks plasmin fibrinolytic activity. In these lysates, plasminogen activator molecules exist as high molecular weight unstable complexes exhibiting a high affinity for plasminogen.
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PMID:Importance, localization and functional properties of the cell-associated form of plasminogen activator in mouse peritoneal macrophages. 668 15

The tissue activator was extracted with 2 M ammonium thiocyanate and purified by L-arginine methyl ester, concanavalin A and ion-exchange chromatographies, and Sephacryl S-200 gel filtration in buffers containing Triton X-100 and/or ammonium thiocyanate. The final preparations had specific activities of 25 000-40 000 IU/mg protein and were shown to be a single band with an apparent molecular weight of 54 00 by SDS-polyacrylamide gel electrophoresis with or without reducing agent. When subjected to isoelectric focusing, its major component had an isoelectric point of approx. 8.2 with minor components. (7.8-8.6). The purified tissue activator was a serine protease, dissimilar to urokinase in some respects including antigenicity, strong affinity to insoluble fibrin monomer and hydrolytic activities for synthetic substrates. The crude extract contained another plasminogen activator with antigen identity to urokinase, which constituted approx. 15% of the total activity in crude extract. These findings indicated that human kidney would produce at least two plasminogen activators, namely, the tissue activator as a major plasminogen activator and urokinase.
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PMID:Purification and characterization of human kidney plasminogen activator dissimilar to urokinase. 705 41

The plasminogen activator content of surgically removed lung cancers, as well as that of adjacent normal lung tissue has been determined quantitatively. Optimum conditions for the quantitative extraction have been worked out using a modification of the technique described by Nagy et al. (Int. J. Cancer, 19:614-620, 1977) which utilizes a buffered solution of the non-ionic detergent Triton X-100. Plasminogen activator was significantly elevated in the tumors (2.5- to 4.3-fold over the normal lung, depending on sample selection and other criteria), although individual variations between tumors were extremely large. No significant correlation was found between the histopathological character of the tumors and the activator content, or between invasiveness and activator content. Using rabbit antibody formed against purified urokinase as an inhibitor of activator action, it was found that lung tumors contain a urokinase-like enzyme as the predominant plasminogen activator, while the activator content of the adjacent normal lung tissue consists of some urokinase-like enzyme, but mostly of an enzyme which is not inhibited by the antibody. The urokinase-like activator has been purified approximately 20,000-fold from lung tumors by the combination of two affinity chromatographic procedures, and was compared with purified urokinase with a molecular weight of 55,000 on all criteria used, the lung tumor activator was identical to urokinase.
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PMID:Content and characterization of plasminogen activators in human lung tumors and normal lung tissue. 719 15


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