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: UMLS:C0272170 (SDS)
50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The development of a simple, sensitive fluorimetric assay for the measurement of cell surface-associated urokinase plasminogen activator (uPA) on viable, adherent HCT116 cells in microtitre plates, after a preincubation with purified human plasminogen is described. The assay determines plasmin activity by the cleavage of H-D-Val-Leu-Lys 4-aminomethyl coumarin under near physiological pH and ionic conditions with a sensitivity in the range of 5-100 mIU uPA/well at excitation 355 nm and emission 460 nm. Plasmin generated during the assay converted all cell-surface sc-uPA to tc-uPA, allowing the determination of total uPA activity. Inhibitor studies (PAI-2, amiloride or Glu-Gly-Arg chloromethylketone) confirmed the specificity of the uPA assay. Removal of these agents prior to assay allowed determination of the cell surface sc-uPA:tc-uPA ratio. Cell surface activity was only partially removed by acid elution. This corresponded with the loss of a number of proteins and uPA-containing species as detected by SDS-PAGE, gelatin enzymography and Western blotting. Although the major protein species eluted had a M(r) of 55 kDa, reacted with a commercial anti-human uPA mAb and correlated with the main lytic zone, other higher M(r) species were also eluted from HCT116 cells. Exogenous uPA increased cell-surface activity markedly on cells previously treated with acid. Following acid elution, cell surface uPA activity was restored after 30h in culture suggesting either de novo synthesis or release of pre-formed uPA with subsequent secretion and binding to uPAR. The assay has enabled studies on adherent cells to address questions about the regulation and expression of cell-surface uPA.
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PMID:Occupancy of the cancer cell urokinase receptor (uPAR): effects of acid elution and exogenous uPA on cell surface urokinase (uPA). 138 63

The relative amounts of immunoreactive plasminogen and active plasmin in different fractions of bovine milk were examined. Raw milk was centrifuged to separate skim, cream, and a somatic cell pellet. Skim milk was centrifuged to separate milk serum and casein micelles. Milk fat globule membranes were isolated from the cream fraction of bovine milk. Proteins from somatic cells were isolated following sonication of the cells. Western blot analysis showed the presence of several forms of plasminogen in bovine milk. The predominant forms of plasminogen identified following electrophoresis under nonreducing conditions were proteins with approximate molecular weights of 88,000, 152,000, and 160,000. The predominant forms of plasminogen identified after electrophoresis under reducing conditions were two proteins with approximate molecular weights of 88,000 and 50,000. The highest amount (82% of the total plasminogen), as determined by an ELISA, was associated with the casein fraction. Lower plasminogen concentrations were associated with the serum, cream fractions, and milk fat globule membranes. The SDS-PAGE of the cream and milk fat globule membranes indicated that some casein was present in both fractions. Thus, the low plasminogen concentrations in these fractions may be associated with the caseins there. No immunoreactive plasminogen was present in the somatic cells. Active plasmin was present in the same milk fractions in which plasminogen was detected: casein, serum, and cream.
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PMID:Distribution of plasminogen and plasmin in fractions of bovine milk. 138 54

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.
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PMID:Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator. 139 99

Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the initial and rate-limiting step in the fibrinolytic cascade, is cleared rapidly in vivo by the liver. Using chemical crosslinking, we have recently identified a plasminogen-activator inhibitor type 1 (PAI-1)-independent t-PA clearance receptor on rat hepatoma MH1C1 cells with a relative molecular mass of approximately 500 kDa. Another recently identified membrane receptor, low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR), was also detected on MH1C1 hepatoma cells by using immunoprecipitation with anti-LRP/alpha 2MR antibody. When analyzed by SDS/PAGE, we found the t-PA receptor identified on MH1C1 cells comigrated with the large subunit of LRP/alpha 2MR. The t-PA receptor was immunoprecipitated by an anti-LRP/alpha 2MR antibody after chemical crosslinking of specifically bound 125I-labeled t-PA to its receptor. Through chemical crosslinking studies, we found that t-PA and methylamine-activated alpha 2-macroglobulin could bind to LRP/alpha 2MR simultaneously without competing with one another for binding, suggesting that the two ligands bound to two independent sites on the LRP/alpha 2MR molecule. Furthermore, a 39-kDa protein, which modulates ligand binding to LRP/alpha 2MR, was also found to inhibit t-PA binding to its receptor. These data thus show that the t-PA clearance receptor identified on MH1C1 hepatoma cells is LRP/alpha 2MR.
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PMID:Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor is an hepatic receptor for tissue-type plasminogen activator. 150 54

Two fibrinolytic enzymes, jararafibrase I and jararafibrase II, were purified from Bothrops jararaca venom. The purified jararafibrase I and jararafibrase II ran as single protein bands on analytical polyacrylamide gel electrophoresis and had mol. wts of 47,000 +/- 2000 and 21,400 +/- 500, respectively, by SDS-polyacrylamide gel electrophoresis. The isoelectric points of jararafibrase I and jararafibrase II were 4.6 and 6.5, respectively. The specific activities of jararafibrase I and jararafibrase II were 2.2 units/mg protein and 6.3 units/mg protein, respectively. Both enzymes exhibited no detectable plasminogen activating activity. The activity of the enzymes was completely inhibited by 1,10-phenanthroline and ethylenediaminetetraacetate, suggesting that both enzymes were metalloproteinases. Jararafibrase I and jararafibrase II had single-chain protein compositions, and the amino acid sequence up to the 49th amino acid from the NH2-terminal of jararafibrase II was: Leu-Pro-Glu-His-Gln-Arg-Tyr-Ile-Glu-Leu-Phe-Ile-Val-Val-Asp-His-Gly-Met- Phe-Met-Lys-Tyr-Asn-Gly-Asn-Ser-Asp-Lys-Ile-Arg-Arg-Arg-Ile-His-Gln- Met-Val-Asn-Ile-Met-Lys-X-Ala-Tyr-Arg-Tyr-Leu-Tyr-Ile-(X = not confirmed).
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PMID:Purification and characterization of two fibrinolytic enzymes from Bothrops jararaca (jararaca) venom. 152 77

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.
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PMID:Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen. 156 67

We examined the effects of polysaccharides on t-PA mediated plasminogen activation using single-chain tissue plasminogen activator (sct-PA) and two-chain tissue plasminogen activator (tct-PA). Unfractionated heparin, low molecular weight heparin (LMW heparin) and dextran sulfates enhanced the activation rate of plasminogen by sct-PA about three-fold to six-fold. Chondroitin sulfate C did not enhance the activation. The activation of plasminogen by tct-PA was slightly enhanced by unfractionated heparin, but not by other polysaccharides. Conversion of sct-PA to tct-PA was not stimulated by polysaccharides. SDS-PAGE showed no enhancement of the conversion from sct-PA to tct-PA by plasmin in the presence of polysaccharides. However, the enhancement of sct-PA mediated activation of plasminogen by unfractionated heparin, LMW heparin and dextran sulfates in the presence of aprotinin was shown with SDS-PAGE. It was suggested that unfractionated heparin, LMW heparin and dextran sulfates form complex with sct-PA and plasminogen, and stimulate the conversion of sct-PA to tct-PA.
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PMID:The effects of polysaccharides on plasminogen activation by single chain-and two chain-tissue plasminogen activator. 165

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.
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PMID:The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of alpha 2-antiplasmin. 169 17

Vitronectin endows plasminogen activator inhibitor 1 (PAI-1), the fast-acting inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), with additional thrombin inhibitory properties. In view of the apparent association between PAI-1 and vitronectin in the endothelial cell matrix (ECM), we analyzed the interaction between PAI-1 and thrombin in this environment. Upon incubating 125I-labeled alpha-thrombin with endothelial cell matrix (ECM), the protease formed SDS-stable complexes exclusively with PAI-1, with subsequent release of these complexes into the supernatant. Vitronectin was required as a cofactor for the association between PAI-1 and thrombin in ECM. Metabolic labeling of endothelial cell proteins, followed by incubation of ECM with t-PA, u-PA, or thrombin, indicated that all three proteases depleted PAI-1 from ECM by complex formation and proteolytic cleavage. Proteolytically inactive thrombin as well as anticoagulant thrombin, i.e., thrombin in complex with its endothelial cell surface receptor thrombomodulin, did not neutralize PAI-1, emphasizing that the procoagulant moiety of thrombin is required for a functional interaction with PAI-1. A physiological implication of our findings may be related to the mutual neutralization of both PAI-1 and thrombin, providing a new link between plasminogen activation and the coagulation system. Evidence is provided that in ECM, procoagulant thrombin may promote plasminogen activator activity by inactivating PAI-1.
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PMID:Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix. 172 12

A hybrid hybridoma (FU1-74), secreting a bispecific monoclonal antibody (bs mAb), was obtained by fusion of a murine hybridoma secreting a monoclonal antibody (mAb) specific for human fibrin with a murine hybridoma secreting a mAb against urokinase-type plasminogen activator (u-PA). The bs mAb (MA-FU1-74), purified to homogeneity from mouse ascitic fluid, migrated as a single band with apparent Mr 150,000 on nonreduced SDS-PAGE and had an affinity for both human fibrin (Ka = 2 x 10(7) M-1) and for u-PA (Ka = 10(8) M-1) comparable to that of the mAbs obtained from the respective parental hybridomas. MA-FU1-74 did not influence the enzymatic activity of two-chain u-PA (tcu-PA) towards plasminogen or towards a chromogenic substrate. The complex of MA-FU1-74 with recombinant single chain u-PA (rscu-PA) or with tcu-PA (urokinase) enhanced the fibrinolytic potency of the plasminogen activator towards clotted human plasma 20-fold and 5-fold, respectively. In a hamster pulmonary embolism model, the rscu-PA/MA-FU1-74 complex had a 13- to 17-fold increased thrombolytic potency (percent lysis per mg/kg u-PA administered) relative to that of rscu-PA. The specific thrombolytic activity (percent lysis per microgram/ml steady state plasma level of u-PA antigen) of the complex was, however, not significantly different from that of rscu-PA. The complex of rscu-PA with the parental anti-u-PA mAb (MA-UK1-3) had only a 2-fold enhanced thrombolytic potency relative to that of rscu-PA and had a 5-fold decreased specific thrombolytic activity. The plasma clearance rates of the complexes of rscu-PA with both MA-FU1-74 and MA-UK1-3 were about 10-fold lower than that of rscu-PA. In a rabbit jugular vein thrombosis model, the rscu-PA/MA-FU1-74 complex had a 4-fold enhanced thrombolytic potency, an unchanged specific thrombolytic activity and 20-fold reduced plasma clearance. In both animal models, the rscu-PA/MA-FU1-74 complex did not cause more extensive systemic activation of the fibrinolytic system than rscu-PA. It is concluded that the bispecific anti-fibrin/anti-u-PA mAb MA-FU1-74 targets u-PA to the fibrin clot, resulting in a significantly enhanced thrombolytic potency of the plasminogen activator.
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PMID:Enhancement of clot lysis in vitro and in vivo with a bispecific monoclonal antibody directed against human fibrin and against urokinase-type plasminogen activator. 179 14


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