Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method is described by which the heavy chain of human plasmin, obtained by partial reduction of urokinase-activated plasminogen with 2-mercaptoethanol, is adsorbed on lysine coupled to polyacrylamide. The heavy chain is recovered from the adsorbent by elution with 6-aminohexanoic acid (yield 60-65%). Sulfhydryl titrations of the heavy chain showed that the partial reduction involved primarily the cleavage of the sole interchain disulfide bridge of plasmin. Dodecylsulfate-polyacrylamide electrophoresis gave essentially a single band corresponding to a component of about 60000 molecular weight. The NH2-terminal amino acid was predominantly threonine. 6-Aminohexanoic acid at different concentrations caused significant variations of the sedimentation and diffusion constants of the heavy chain indicating inhibitor-induced conformational alterations of the protein. The present results suggest that in plasmin only the heavy chain is capable of interacting with 6-aminohexanoic acid, and it appears that it is primarily this chain which plays an important role in the inhibition of the enzyme by 6-aminohexanoic acid.
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PMID:A new method of isolation and some properties of the heavy chain of human plasmin. 12 54

The preparation of a new class of covalent hybrid plasminogen activators containing the fibrin-binding domains of human plasmin(ogen) and the catalytic active center of human urokinase will be described. Hybridization of the sulfhydryl form of the NH2-terminal plasmin-derived heavy (A) chain (PlnA) with the sulfhydryl form of the COOH-terminal urokinase-derived active heavy (B) chain (u-PAB) was carried out; a covalent PlnA-u-PAB hybrid plasminogen activator was prepared. The sulfhydryl form of PlnA (PlnA(SH)2) was isolated from reduced Lys-2-plasmin by L-lysine-substituted Sepharose column chromatography. For the isolation of the sulfhydryl form of u-PAB (u-PAB(SH], high molecular weight urokinase was adsorbed onto a benzamidine-Sepharose column and reduced with 100 mM 2-mercaptoethanol on the column. The urokinase NH2-terminal light (A) chain was washed off the column, and the u-PAB(SH) chain was eluted from the column. The specific activity of the isolated u-PAB(SH) chain was determined to be 242 000 IU/mg of protein. The PlnA(SH)2 and u-PAB(SH) chains were mixed at a molar ratio of PlnA(SH)2 to u-PAB(SH) of 3:2; the reducing agents were then removed by gel filtration. The hybridization (reoxidation) reaction was allowed to proceed for 48 h at 4 degrees C. The covalent hybrid activator, in 40% yield, was purified from the reaction mixture to homogeneity, by a sequential affinity chromatography method with L-lysine-substituted Sepharose followed by anti-low molecular weight urokinase IgG-Sepharose, and then gel filtration through Sephadex G-150.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Covalent molecular weight approximately 92 000 hybrid plasminogen activator derived from human plasmin amino-terminal and urokinase carboxyl-terminal domains. 294 Oct 75

Treatment of bovine pulmonary arterial and aortic endothelial cells for 24 h with 10-20 ng/ml endotoxin appeared to suppress urokinase secretion by over 90%. Immunodepletion of urokinase from conditioned medium revealed high levels of a fully active plasminogen activator inhibitor (PAI). In contrast, medium from aortic cells not treated with endotoxin expressed low levels of a latent PAI. The endotoxin-induced PAI from both cell types was not immunoprecipitated by incubation with IgG directed against the endothelial-type inhibitor, PAI-1, or antiserum directed against the placental-type inhibitor, PAI-2. Endotoxin-induced PAI was inactivated by 0.2-1.0 M of 2-mercaptoethanol, but unaffected by treatment with up to 500 microM of chloramine-T. We conclude that the exposure of bovine endothelial cells to endotoxin causes the secretion of a fully active PAI with properties different from those reported for PAI-1.
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PMID:Endotoxin-induced secretion of an active plasminogen activator inhibitor from bovine pulmonary arterial and aortic endothelial cells. 312 Jul 14

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.
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PMID:Effect of dithiothreitol on activity and protein structure of human urine urokinase. 399 96

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

A factor, named SK-potentiator, which is known to potentiate the activation of plasminogen by streptokinase (SK), was isolated from human plasma. The procedures consisted of column chromatographies on DEAE-Sephadex A-50 and heparin-agarose, followed with gel-filtration on a Sepharose 6B column and affinity chromatography on a lysine-Sepharose 4B column. The isolated SK-potentiator markedly potentiated the rate of activation of plasminogen by streptokinase. However, it did not show a potentiating effect on the activation of plasminogen by urokinase or on the plasmin activity. SK-potentiator showed a similar mobility to that of fibrinogen on both SDS-polyacrylamide gel and agarose gel electrophoresis, and cross-reacted with anti-fibrinogen antiserum. The amino acid composition of SK-potentiator was very close to that of human fibrinogen, although the content of serine and threonine was significantly lower. SK-potentiator showed a single band with a molecular weight of 300,000 on SDS-gel electrophoresis in the absence of 2-mercaptoethanol. In the presence of 2-mercaptoethanol, it showed two major bands with molecular weights of 53,000 and 48,000, respectively, which corresponded to the B beta chain and gamma chain of fibrinogen. To establish further that the isolated SK-potentiator may be one of the fibrinogen degradation products (FDP), human fibrinogen was digested with plasmin and the SK-potentiator activity generated in the course of the digestion was measured. As a result, the SK-potentiator activity was found to initially increase and then decrease with incubation time, suggesting that an early FDP has the ability to potentiate the SK-mediated activation of plasminogen. The early FDP was then isolated by gel-filtration on a column of Sepharose 6B and it was found that the SK-potentiator activity was associated with the early FDP. Moreover, the early FDP showed the same electrophoretic mobility as the isolated SK-potentiator on SDS-gel electrophoresis and their amino acid compositions were quite similar to each other. From these results, the SK-potentiator was identified as the early FDP.
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PMID:Streptokinase-dependent potentiating factor (SK-potentiator) for plasminogen activation from human plasma: its identification as a fibrinogen degradation product. 689 Sep 55

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.
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PMID:A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea. 1066 46

Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency but didn't show any reactivity for the gamma form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30 degrees C and the maximum fibrinolytic activity was at 55 degrees C.
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PMID:Characterization of a metalloenzyme from a wild mushroom, Tricholoma saponaceum. 1130 69