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)

Two plasminogen activators (1 and 2) were isolated from human seminal plasma by hiigh-speed centrifugation, Sephadex-gel filtration and ion-exchange chromatography. The activators were shown to be homogeneous by polyacrylamide-disc -gel electrophoresis at pH 8.3 and 4.5, and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights of activators 1 and 2 were estimated as 69 000 and 74 000. Their amino acid compositions are very similar, both being high in aspartic acid, glutamic acid, serine, glycine and leucine, and low in methionine, tryptophan, tyrosine, isoleucine and histidine. Activators 1 and 2 each possess 16 cysteine residues. Both activators have isoelectric points of approx. 7.0, are stable over a wide pH range at temperatures up to 60 degrees C, but lose activity at higher temperatures, particularly under very basic or acidic conditions. They are not inhibited by EDTA, Mg2+ and Ca2+ at 10 mM concentrations, but their activity decreases on addition of 10 mM-cysteine or Fe2+ and 6-aminohexanoate or sera from pregnant women. The precipitin band formed between urokinase and its antiserum is continuous with the precipitin bands formed between the seminal plasminogen activators and the urokinase antiserum. Antisera to urokinase inhibit both the activity of urokinase and the seminal plasminogen activators.
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PMID:Purification of plasminogen activators from human seminal plasma. 2 36

A cDNA library derived from human carcinoma cells was used to isolate a clone, pULB1000, coding for the preproenzyme form of human urokinase. This clone carries the full-length sequence coding for the signal peptide and for the A chain (157 amino acids) and B chain (253 amino acids) of urokinase in tandem. The sequence of the cDNA predicts the presence of a single lysine residue between the last amino acid of the mature A polypeptide (Phe-157) and the first amino acid of the mature B polypeptide (Ile-1). The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence data with three exceptions, the reported cysteine residue at position 131 in the A chain is a tryptophan, and glycine 366 and alanine 410 in the B chain are, respectively, a cysteine and a valine in our clone. A large Bgl I fragment (1482 bp), derived from the clone pULB1000 coding for most of the signal peptide and for the A and B chains, has been subcloned into the expression vector pCQV2. Heat induction of E. coli cells carrying the recombinant plasmid leads to the production of urokinase-like polypeptides having the expected molecular weights and being specifically recognized by antibodies raised against natural human urokinase.
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PMID:Molecular cloning, sequencing, and expression in Escherichia coli of human preprourokinase cDNA. 388 71

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.
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PMID:The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain. 675 69

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.
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PMID:Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium. 963 82

Four novel triprenyl phenol metabolites, designated SMTP-3, -4, -5, and -6, have been isolated from cultures of Stachybotrys microspora IFO 30018 by solvent extraction and successive chromatographic fractionation using silica gel and silica ODS columns. A combination of spectroscopic analyses showed that SMTP-3, -4, -5, and -6 are staplabin analogs, containing a serine, a phenylalanine, a leucine or a tryptophan moiety in respective molecules in place of the N-carboxybutyl portion of the staplabin molecule. SMTP-4, -5, and -6 were active at 0.15 to 0.3 mM in enhancing urokinase-catalyzed plasminogen activation and plasminogen binding to fibrin, as well as plasminogen- and urokinase-mediated fibrinolysis. On the other hand, the concentration of staplabin required to exert such effects was 0.4 to 0.6 mM, and SMTP-3 was inactive at concentrations up to 0.45 mM.
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PMID:Isolation of SMTP-3, 4, 5 and -6, novel analogs of staplabin, and their effects on plasminogen activation and fibrinolysis. 1004 63

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.
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PMID:Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms. 1050 32

The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.
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PMID:Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen. 1089 32

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.
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PMID:Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein. 1141 62

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.
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PMID:A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition. 1235

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55 degrees C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), beta-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bbeta-chains and Aalpha-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.
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PMID:Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. 1657 57


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