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)

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

Investigations are reported on the composition of protein layers adsorbed from plasma to various modified polystyrene resins. As well as polystyrene itself, polystyrene bearing sulfonate groups in the benzene rings, and polystyrene sulfonate in which the sulfonate groups were converted to amino acid sulfamide, were investigated. Some of these resins were shown in previous work to have anticoagulant properties. To study the adsorption of proteins from plasma, the resins were exposed to citrate anticoagulated human plasma for 3 h. Adsorbed proteins were then eluted sequentially by 1M Tris buffer and 4% SDS solution, and examined by SDS-PAGE. The gel patterns were similar on all resins except polystyrene. From the MWs of the gel bands, the major protein component appeared to be fibrinogen. Smaller amounts of plasminogen, transferrin, albumin, and IgG were also present. In addition, Ouchterlony immunoassay of the eluates from one resin gave positive identification of complement C3, fibronectin, IgG, and IgM. Many other minor gel bands remain unidentified. A consistent finding for all resins was the presence of plasmin-type fibrinogen degradation products though the amounts varied with resin type. It is concluded from this (and from experiments showing FDP formation when fibrinogen was absorbed to the resins, from buffer containing a trace of plasminogen) that the functional groups in these materials promote the adsorption of plasminogen and its activation to a plasmin-like molecule. It appears from the substantial quantities of fibrinogen adsorbed to these materials after 3 h exposure to plasma that the Vroman effect (giving transient adsorption of fibrinogen) is not operative on these materials. It is hypothesized that specific interactions occur between fibrinogen and sulfonate groups.
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PMID:Interactions of proteins in human plasma with modified polystyrene resins. 201 12

Various purified proteins, protein derivatives and two polysaccharides were added individually to a physiological medium in order to effect uptake of 125I-thrombin by the rabbit aorta endothelium. Over a wide range of concentration (0.004-40 mg/ml), the presence of either purified rabbit or bovine albumin during thrombin uptake encouraged an increase (70-110%) in 125I-thrombin binding by the endothelium and subendothelium compared to uptake by aorta segments in the absence of added protein. Pretreatment of aorta segments with albumin before incubation with 125I-thrombin in the absence of albumin did not encourage thrombin uptake to the same extent as having 125I-thrombin and albumin together. Purified human transferrin, rabbit IgG, chicken ovalbumin or denatured bovine casein could replace albumin to produce a similar enhancement of thrombin uptake. Replacing active concentrations of albumin by either reduced-carboxymethylated albumin, defatted albumin, plasmin-treated or thermolysin-treated albumin also caused an increase (50-130%) in thrombin binding, whereas replacement by acid-hydrolysed albumin or with polyglutamic acid was either ineffective or even inhibitory. Lysine-modified or arginine-modified albumins caused a small enhancement (14-32%) and no enhancement of thrombin uptake, respectively. Dextran, at low concentration (0.04-0.4 mg/ml) did not influence thrombin uptake, and at higher concentration (4-40 mg/ml) caused a decrease in uptake by both the endothelium and subendothelial layers. Low concentration of dextran sulphate inhibited thrombin uptake to 20-30% of control values. These data express the importance of accompanying protein in the response of the vascular endothelium during binding of thrombin. The possibility that other protein-cell interactions may be similarly influenced by macromolecular solutes is also discussed.
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PMID:The presence of plasma proteins facilitates the uptake of 125I-thrombin by the rabbit thoracic aorta endothelium in vitro. 242 49

Human transferrin, alpha 2-macroglobulin, and fibrinogen were incubated with [3H]-glucose. After a 7-day, 37 degrees C incubation at 20 mM glucose, transferrin incorporated 1.1 mol of glucose/mol protein; alpha 2-macroglobulin, 10 mol of glucose/mol; and fibrinogen, 3.8 of glucose/mol, or approximately 14 mumol of glucose/g for each protein. These results were the same for glucose labeled in the 1 or 6 position. No radiolabel was incorporated into the proteins during incubations with glucose labeled in the 2 position. The rate and extent of iron binding were identical for both glucosylated and nonglucosylated transferrin. Glucosylated transferrin bound to Wil-2 human lymphoblast cells with a Kd = 33 nM and receptor number of 3.4 X 10(5) receptors/cell; nonglucosylated transferrin with a Kd = 31 nM and receptor number of 3.9 X 10(5) receptors/cell. Glucosylated and nonglucosylated alpha 2-macroglobulin showed the same conformational change as determined on native PAGE after reaction with trypsin, plasmin, or methylamine and had the same activity in the Ganrot assay after reaction with trypsin or plasmin. The clearance of 125I-labeled, methylamine-treated alpha 2-macroglobulin from the mouse circulation was identical for both glucosylated and nonglucosylated alpha 2-macroglobulin, t1/2 = 3 min. alpha 2-Macroglobulin that was first glucosylated then reacted with methylamine bound to mouse peritoneal macrophages with a Kd of 2.5 nM and receptor activity of 370 fmol/mg cell protein. alpha 2-Macroglobulin that was first reacted with methylamine and then glucosylated bound with a Kd of 3 nM and receptor activity of 320 fmol/mg cell protein. Glucosylated fibrinogen had the same clotting time as control fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro preparation of nonenzymatically glucosylated human transferrin, alpha 2-macroglobulin, and fibrinogen with preservation of function. 258 Jul 49

The effect of retinoic acid on the synthesis and degradation of basement membrane components by endoderm cells derived from mouse embryonal carcinoma (EC) cells was studied in a serum-free, defined medium. By immunofluorescence these cells accumulate type IV collagen, laminin, and fibronectin after growth in media containing epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin, transferrin, and Pedersen fetuin. Collagen accounted for 2 to 4% of the newly synthesized proteins, of which 90% were found in the culture media. This collagen was identified as Pro-type IV be gel electrophoresis and enzymatic susceptibility. The EC cells preferentially attached to type IV collagen in vitro and such attachment was mediated by laminin. Treatment of EC cells with retinoic acid caused an increased accumulation of collagen (10 to 15% of secreted proteins) and also stimulated the elaboration of latent protease which degraded laminin and type IV collagen. The laminin-degrading activity was plasminogen dependent. The type IV collagen-degrading activity was a metal protease which could be activated by trypsin or plasmin. It is likely that at least part of the laminin degrading activity is plasmin (mediated through plasminogen activator), since highly purified plasmin is shown to degrade native laminin.
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PMID:Stimulation of retinoic acid of synthesis and turnover of basement membrane in mouse embryonal carcinoma-derived endoderm cells. 628 41

In view of conflicting evidence for a major hepatic role in the synthesis of circulating plasminogen, the precursor of the fibrinolytic enzyme plasmin, we carried out the present study with a sensitive assay in order to measure the accumulation of small quantities of plasminogen in a recycling rat liver perfusion system. We have purified plasminogen from Sprague-Dawley rat plasma and have raised a monospecific antiserum against it in rabbits. Isolated rat liver perfusions were performed with an oxygenated recycling perfusate consisting of a perfluorotributylamine/Pluronic F-68 emulsion (Fluosol 43) free of plasma proteins and blood cells. The system was shown to be capable of synthesizing albumin and transferrin. The cumulative appearance of plasminogen in the perfusate was measured by a sensitive, specific radioimmunoassay. Plasminogen concentration increased progressively during the first 2 hr of perfusion; the observed average net synthesis in five separate experiments was approximately 35 microgram/hr per 100 g of body weight. Exposure of the perfused liver to 18 microM cycloheximide inhibited additional increase in the titer of plasminogen. Evidence for de novo synthesis was provided by the incorporation of 14C-labeled leucine into specific immunoprecipitates of plasminogen and the inhibition of this incorporation by cycloheximide. Analysis of the immunoprecipitates by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a single peak of radioactivity corresponding to Mr of 82,000. These data indicate that the liver is a major site of plasminogen production.
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PMID:Production and release of plasminogen by isolated perfused rat liver. 693 86

The authors sought to evaluate the pathogenetic and prognostic role of a procoagulant and hypofibrinolytic state in the adult respiratory distress syndrome (ARDS). Twenty-two consecutive patients admitted to the intensive care unit (ICU) for respiratory monitoring (n = 2) or mechanical ventilation (n = 20) were studied, of whom 13 had ARDS and 9 were at risk for the syndrome. Plasma levels of thrombin-antithrombin III complexes (TAT), the plasmin-alpha2-antiplasmin complexes (PAP), tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) were measured within 48 h after admission, together with respiratory variables allowing computation of the lung injury score (LIS), and pulmonary microvascular permeability [67Gallium-transferrin pulmonary leak index (PLI)], as measures of pulmonary dysfunction. Blood was also sampled 6-hourly until 2 days after admission. The LIS and PLI were higher in ARDS than at risk patients, in the presence of similar systemic morbidity and mortality. TAT complexes were elevated in a minority of patients of both groups, whereas the PAP, tPA and PAI levels were elevated above normal in the majority of ARDS and at risk patients, but groups did not differ. Neither circulating coagulation nor fibrinolysis variables correlated to either LIS or PLI. Furthermore, the course of haemostatic variables did not relate to outcome. These data indicate that systemic activation of coagulation and impaired fibrinolysis do not play a major role in ARDS development and outcome in patients with acute lung injury.
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PMID:Systemic coagulation and fibrinolysis in patients with or at risk for the adult respiratory distress syndrome. 942 92

Staphylococcus aureus and Staphylococcus epidermidis possess a 42-kDa cell wall transferrin-binding protein (Tpn) which is involved in the acquisition of transferrin-bound iron. To characterize this protein further, cell wall fractions were subjected to two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis blotted, and the N-terminus of Tpn was sequenced. Comparison of the first 20 amino acid residues of Tpn with the protein databases revealed a high degree of homology to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Analysis of staphylococcal cell wall fractions for GAPDH activity confirmed the presence of a functional enzyme which, like Tpn, is regulated by the availability of iron in the growth medium. To determine whether Tpn is responsible for this GAPDH activity, it was affinity purified with NAD+ agarose. Both S. epidermidis and S. aureus Tpn catalyzed the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. In contrast, Staphylococcus saprophyticus, which lacks a Tpn, has no cell wall-associated GAPDH activity. Native polyacrylamide gel electrophoresis of the affinity-purified Tpn revealed that it was present in the cell wall as a tetramer, consistent with the structures of all known cytoplasmic GAPDHs. Furthermore, the affinity-purified Tpn retained its ability to bind human transferrin both in its native tetrameric and SDS-denatured monomeric forms. Apart from interacting with human transferrin, Tpn, in common with the group A streptococcal cell wall GAPDH, binds human plasmin. Tpn-bound plasmin is enzymatically active and therefore may contribute to the ability of staphylococci to penetrate tissues during infections. These studies demonstrate that the staphylococcal transferrin receptor protein, Tpn, is a multifunctional cell wall GAPDH.
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PMID:The staphylococcal transferrin-binding protein is a cell wall glyceraldehyde-3-phosphate dehydrogenase. 1002 47

Fibrinogen (FBG), together with its polymerized form fibrin, modulates cellular responses during wound repair and tissue remodeling. Thus, we sought to determine whether A549 lung epithelial type II-like cells would endocytose insoluble, surface-bound FBG as a potential mechanism of alveolar matrix remodeling. Surface-bound FBG was endocytosed into either lysosomes or late endosomes by A549 cells through arg-gly-asp-dependent binding to alphavbeta3 but not alpha5beta1 integrin receptors. Soluble FBG added to confluent monolayers of A549 cells was not endocytosed. Unlike the uptake of the extracellular matrix glycoproteins vitronectin and thrombospondin by other cell types, endocytosis of FBG by A549 cells was neither inhibited by heparin nor dependent on binding to cell-surface heparan sulfate proteoglycans. FBG did not colocalize with endocytosed transferrin, whereas dextran showed partial colocalization with FBG in endocytic vesicles, suggesting nonclathrin-mediated endocytosis. Inhibition of actin filament polymerization blocked endocytosis of both dextran and FBG but not transferrin, providing further support that FBG is endocytosed via a nonclathrin pathway. Disruption of actin polymerization inhibited integrin-mediated cell spreading, which contributed to an overall reduction in FBG clearance that was most likely due to reduced cell migration and associated pericellular proteolysis. Trasylol inhibition of extracellular plasmin activity did not inhibit endocytosis of FBG. The endocytosed FBG was degraded to trichloroacetic acid-soluble fragments that showed an electrophoretic pattern distinctly different from plasmin-degraded FBG. Together, these results suggest that endocytosis of matrix-associated FBG by alveolar epithelial cells may be involved in the processes of alveolar tissue repair and matrix remodeling.
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PMID:Integrin alphavbeta3-mediated endocytosis of immobilized fibrinogen by A549 lung alveolar epithelial cells. 1115 45

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