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 cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand interaction between urokinase-type plasminogen activator and its receptor probed with 8-anilino-1-naphthalenesulfonate. Evidence for a hydrophobic binding site exposed only on the intact receptor. 804 85

Tegumental extracts from adult worms of Schistosoma mansoni contain an inhibitory activity to the S. mansoni 28-kDa serine protease and to pancreatic elastase. By using biotinylated elastase and streptavidin-agarose, the postulated protease inhibitor has been isolated from the crude worm extract in a single step. Monospecific rabbit antibodies raised against the protease inhibitor have immunoprecipitated a 56-kDa [35S]Met-labeled serine protease inhibitor which was designated Smpi56 (S. mansoni protease inhibitor, 56 kDa). Smpi56 binds tightly to and inhibits the 28-kDa protease of S. mansoni and pancreatic and neutrophil elastase but not papain, pepsin, thrombin, trypsin, chymotrypsin, proteinase K, urokinase and acetylcholinesterase. The biological function of Smpi56 is still not known, but in view of its elastase inhibitory activity it may be speculated that the parasite is employing Smpi56 to protect itself from activated neutrophils. Smpi56 may also potentially protect the parasite from its endogenous 28-kDa protease.
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PMID:Schistosoma mansoni: isolation and characterization of Smpi56, a novel serine protease inhibitor. 811 69

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.
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PMID:Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases. 834 6

The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.
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PMID:Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. 839 46

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).
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PMID:Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect. 861 81

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.
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PMID:Enhancement of catalytic activities of serine proteases by tripeptides compounds. 863 1

Human cytoplasmic antiproteinase (CAP) is an intracellular serpin that has been reported to utilize Arg341 as the reactive site P1 residue to neutralize a broad variety of extracellular serine proteases with trypsin-like specificity. Both native CAP and recombinant CAP purified from Escherichia coli were observed to form SDS-stable complexes not only with 125I-thrombin and 125I-urokinase, but also with 125I-chymotrypsin. Kinetic studies indicated that the amidolytic activity of chymotrypsin is inhibited efficiently and rapidly by CAP in a two-step process with a dissociation constant Ki of an initial loose complex of 3.3 nM, a forward isomerization rate constant k2 to the tight complex of 0.014 s-1, and an overall second order association rate constant of 6 x 10(6) M-1 s-1, similar to the kinetic constants obtained for the formation of the trypsin-CAP complex. N-terminal amino acid sequencing and mass spectrometry indicated that chymotrypsin interacts with CAP at Met340, in contrast to thrombin, which interacts as expected at Arg341. Thus, CAP is the first serpin that has been shown to be capable to inhibit efficiently and with similar association rate constants different proteases at distinct reactive site residues, strongly supporting the notion of a highly mobile and flexible serpin reactive site loop and suggesting that this inhibitor may have evolved separate reactive sites for the specific regulation of different proteolytic activities.
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PMID:Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteases utilizing distinct reactive site residues. 866 39

A low molecular mass monomeric protein termed Fh-KTM (Fasciola hepatica Kunitz-type molecule) was isolated from the trematode Fasciola hepatica. Fh-KTM is a single polypeptide of 58 amino acids and a Mr of 6751. The complete amino acid sequence of Fh-KTM was determined and revealed significant similarity to the Kunitz-type (BPTI) family of proteinase inhibitors. Several polymorphisms were observed suggesting that more than one Fh-KTM molecule may be expressed by this parasite. Modified proline residues were shown to occur at all four positions in this protein as 3-hydroxy derivatives. This is the first report of 3-hydroxyproline residues in a Kunitz-type molecule. Indirect immunofluorescence and immunogold labelling revealed that Fh-KTM is an abundant molecule within the parasite localised to the gut, the parenchymal tissue and the tegument of adult F. hepatica. Serine protease inhibition assays revealed that Fh-KTM exhibited little or no inhibition against chymotrypsin, kallikrein, urokinase or key serine proteases of the blood coagulation pathways. However, Fh-KTM was able to inhibit trypsin even though the P1 reactive amino acid of Fh-KTM was a leucine residue.
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PMID:Characterisation of a novel Kunitz-type molecule from the trematode Fasciola hepatica. 871 42

The interaction of novel series of synthetic inhibitors with various serine proteases (leukocyte elastase, thrombin, cathepsin G, chymotrypsin, plasminogen activators and plasmin) and an aspartic protease (HIV-1 protease) were studied. Various aspects were analyzed: mechanism of action, structure-activity relationships, and in some cases, molecular modelling and biological evaluation. Functionalized cyclopeptides and N-aryl azetidin-2-ones behaved as suicide substrates acting specifically on trypsin-like proteases (thrombin or urokinase) and elastases, respectively. Novel hydrazinopeptides acted as reversible inhibitors of elastases. Coumarin derivatives inactivated very efficiently chymotrypsin-like proteases (k(inact)/K(I) = 760,000 M(-1) .s(-1)). Inhibitors of HIV-1 protease acting either as inactivators or dimerization inhibitors are under investigation. The inhibitors described above are useful for elucidating the biological roles of the target enzymes and constitute potential drugs.
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PMID:[Synthetic inhibitors targeting serine and aspartic acid proteases]. 877 49


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