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:3.4.21.73 (urokinase-type plasminogen activator)
10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The levels of fibrinogen and of profibrinolysin (plasminogen) in urokinase-treated plasma as a function of time of incubation were measured. The profibrinolysin concentration was estimated through its complete conversion to fibrinolysin and the inhibition of the enzyme by crystalline soybean trypsin inhibitor. The dissociation constant of the FL-STI complex was determined to be 7 times 10-9 M. The average concentration of profibrinolysin in normal human citrated plasma was found to be 8 times 10-7 M. From the decrease of fibrinogen with time in the urokinase-treated plasma, the free fibrinolysin was calculated. Free fibrinolysin in normal human blood in vivo was estimated from the half-life of fibrinogen and other data obtained in this study to be present at a concentration of 1.7 times 10-10 M. The plasmakinase activity in vivo, expressed as urokinase molarity, is also about 2 times 10-10 M.
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PMID:Molar concentrations of fibrinolytic components, especially free fibrinolysin, in vivo. 12 74

When human plasminogen (Glu-Pga) is activated by urokinase in the presence of pancreatic trypsin inhibitor, the plasmin produced (Glu-Pma) exclusively contains a heavy chain (Glu-Ha) derived intact from the original NH2 terminus of Glu-Pga. Similar activations, utilizing a low molecular weight synthetic plasmin acylating agent, p-nitrophenyl-p-(pyridiniummethyl) benzoate, still result in a plasmin molecule with approximately 50% of the plasmin heavy chain containing the intact NH2 terminus of the original Glu-Pga. Activations performed at high levels of urokinase in the absence of any inhibitors initially produce Glu-Pma. However, the final stable plasmin, Lys-Pmb, which is obtained contains a heavy chain (Lys-Hb) which arises by plasminolysis of a small peptide from the NH2 terminus of Glu-Ha. Alternatively, Lys-Pmb can be formed in a separate series of reactions initially involving plasminolysis of Glu-Pga to yield Lys-Pgb. The peptide removed in this step is identical to the peptide removed in the Glu-Ha to Lys-Hb reaction. Next, urokinase catalyzes the conversion of Lys-Pgb to Lys-Pmb without further loss of peptide material. This latter pathway involving Lys-Pgb is probably the major pathway for human Lys-Pmb generation. These studies support a mechanism of activation of human plasminogen which involves at least two bond cleavages in Glu-Pga. However, these same studies strongly indicate that the Nh2-terminal peptide need not be released from Glu-Pga prior to plasmin formation. Further, we feel that plasmin and not urokinase catalyzes cleavage of the NH2-terminal peptide bond from Glu-Pga and the Glu-Ha heavy chain of Glu-Pma.
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PMID:Mechanism of the urokinase-catalyzed activation of human plasminogen. 13 42

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

1. An activator of leucocyte latent collagenase has been extracted from rheumatoid synovial fluid by a preparative method consisting of six steps including precipitation by ammonium sulphate and chromatography on Sephadex G-100, QAE-Sephadex and SP-Sephadex C-50. The purification factor was nearly 1000 and the activator isolated could be shown to have a high degree of homogeneity.--2. Gel chromatography indicated a molecular weight of ca. 60 000.--3. Kinetic studies of the activation and inactivation of the activator during incubation at higher temperatures demonstrated its enzymic nature.--4. Activation of latent collagenase was partially inhibited by iPr2P-F and KCN. Soybean trypsin inhibitor, iodoacetamide, TosLysCH2Cl and TosPheCH2Cl had no effect.--5. Leucocyte latent collagenase was also activated by an excess of trypsin and p-hydroxymercuribenzenesulphonic acid, but only to the extent of about 40% of its activation capacity. Purified neutral protease from human leucocyte granules had no effect on latent collagenase.--6. Several typical substrates for proteases, peptidases, esterases and glycosidases were not attacked by the activator. The possibility that the activator is a known enzyme, such as kallikrein, urokinase or cathepsin B1, could be excluded.
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PMID:Purification and some properties of collagenase proenzyme activator from rheumatoid synovial fluid. 21 83

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin >> human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study. 129 2

A novel model of arterial thrombosis was developed. A mechanical endothelium-denuding injury was created (using a scalpel blade) on harvested, freezer-stored rat carotid arteries. Vessel length of 5 mm. were grafted into the femoral arteries of recipient Sprague-Dawley rats using microvascular anastomotic technique. Patency rates in untreated animals were compared with those in animals receiving systemic aspirin or heparin. The control group patency after 2 hours of flow was 15%, while grafts in aspirin- and heparin-treated animals achieved 35% and 95% patency rates, respectively. Uninjured non-frozen carotid grafts in untreated animals yielded a 95% patency rate, while frozen grafts achieved an 80% patency. Therapeutic levels of aspirin, heparin, and urokinase were confirmed through tail bleeding and whole blood clotting tests, as well as platelet aggregation studies and scanning electron microscopy of the graft lumenal surfaces. A long-term series using syngeneic grafts placed in recipients (Lewis-to-Lewis) and employing systemic heparinization demonstrated maintenance of patency for 4 weeks. Scanning electron microscopy revealed good re-endothelialization, well advanced by one week. Histology confirmed the regrowth of endothelial cells, but showed sparse cellular repopulation of medial and adventitial layers. The mechanical injury model was compared to enzymatic de-endothelialization (using trypsin or collagenase), for which patency rates were similar (10% and 0%, respectively). Trypsin de-endothelialized vessels were tested in vitro for the amount of active trypsin remaining bound to the lumenal surface; no detectable activity was found when trypsin inhibitor was applied following trypsin treatment. The versatility of allowing both in vitro evaluation and in vivo patency assessment demonstrates the uniqueness and value of this new model, offering an avenue toward more direct investigations of surface-mediated thrombotic processes.
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PMID:The de-endothelialized rat carotid arterial graft: a versatile experimental model for the investigation of arterial thrombosis. 144 May 9

Protease nexin I (PNI), a 43,000- to 50,000-dalton glycoprotein, is a potent thrombin and urokinase inhibitor produced by many mammalian cells, including human glia, in tissue culture. PNI is a member of the growing superfamily of serine protease inhibitors now known as serpins, but, unlike many others of this family, it has not yet been detected in normal human plasma. Of interest to neurobiology and neurologic disease, PNI is identical to a glia-derived neurite-promoting factor, glia-derived nexin (GDN). Antibody to PNI stains the periphery of senile amyloid plaques in brain tissue from patients with Alzheimer's disease (AD), along with another serpin, alpha 1-antichymotrypsin (alpha 1-ACT). A soluble form of the beta-amyloid precursor protein (beta APP), containing a Kunitz-type trypsin inhibitor domain, the beta APP751 form, is identical to protease nexin II (PNII), a 100,000-dalton serine protease inhibitor present in a number of tissues besides the brain. PNII/beta APP is also found in normal and AD CSF. We found a 47,000-dalton PNI, a thrombin- and urokinase-inhibiting serpin, in normal human CSF by Western blotting using a monospecific antibody. We also demonstrated biologically active PNI capable of forming complexes with serine proteases 125I-urokinase or 125I-thrombin.
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PMID:Protease nexin I, thrombin- and urokinase-inhibiting serpin, concentrated in normal human cerebrospinal fluid. 162 Mar 46

Induction of HLA-DR antigen expression by interferon-gamma (IFN-gamma) is inhibited by trypsin inhibitors and an anti-trypsin monoclonal antibody, but not by chymotrypsin inhibitors, suggesting a requirement for trypsin-like protease (TLP) activity in IFN-gamma-induced HLA-DR expression. Using p-nitroanilide and thioester substrates, TLP activity was demonstrated in cellular extracts of a hybrid epidermal cell line and judged to be essential for HLA-DR expression. TLP activity was inhibited by the trypsin inhibitors soybean trypsin inhibitor, ovomucoid trypsin inhibitor, and tosyl-lysyl-chloromethyl ketone and by an anti-trypsin monoclonal antibody, closely paralleling inhibition of HLA-DR expression by such agents. TLP activity was enhanced by exposure to trypsin-linked agarose, indicating that the protease normally exists in an inactive form, perhaps in an enzyme-inhibitor complex or as an activatable proenzyme. Finding glucocorticoids (GC) to also inhibit IFN-gamma-induced HLA-DR expression and to regulate serine protease, especially urokinase plasminogen activator (uPA), activity raised the possibility of GC regulation of TLP activity. However, TLP activity was found to be constitutively expressed, regulated by neither GC nor IFN-gamma, nor was uPA activity involved in HLA-DR regulation. Trypsin inhibitors and GC also inhibited induction of intracellular 2',5'-oligoadenylate (2-5A) synthetase by IFN-gamma. Thus, TLP activity is required for IFN-gamma induction of HLA-DR and 2-5A synthetase.
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PMID:Induction of HLA-DR by interferon-gamma requires a trypsin-like protease. 177 67

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.
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PMID:Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous. 191 44

A new cell line (LC-1/sq) of human lung squamous-cell carcinoma was established from a surgically resected specimen of primary lung cancer. Upon continuous propagation in serum-free culture medium, it secreted trypsin inhibitors into the conditioned medium. The major fraction of the trypsin inhibitor (T1-1) was purified to apparent homogeneity by anion-exchange and gel-filtration high-performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transblotting to Immobilon. T1-1 effectively inhibited trypsin. Chymotrypsin, plasmin and kallikrein were inhibited to a lesser extent, but urokinase-type plasminogen activator, elastase, thrombin and papain were not inhibited. The activity of T1-1 was acid-stable and heat-resistant, and its molecular weight was 115 kDa by SDS-PAGE. It exhibited single NH2-terminal sequence, and its first 20 NH2-terminal amino-acid residues were identical with those of protease nexin-II (PN-II)/amyloid beta-protein precursor (APP). These characteristics of T1-1 suggest that the major trypsin inhibitor secreted by LC-1/sq is indistinguishable from PN-II/APP. LC-1/sq is the first lung squamous carcinoma cell line that secretes functionally active trypsin inhibitor, PN-II/APP, in vitro and is useful for studying its biological significance in malignant tumor.
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PMID:Establishment of a new human cancer cell line secreting protease nexin-II/amyloid beta protein precursor derived from squamous-cell carcinoma of lung. 191 42


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