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)

Several specific inhibitors for plasminogen activators have been isolated from various organs and cell lines, those from human placenta and the human monocyte-like cell line U-937 being virtually identical. The reaction between this type of inhibitor, designated as type-2, and high-Mr and low-Mr urokinase-type plasminogen activators was followed by reversed-phase high-performance liquid chromatography and gel electrophoresis. The components, their stable complexes and their dissociation and cleavage products could be clearly identified in both systems. The amino acid sequence of the inhibitor at the cleavage site was determined to be -Met-Thr-Gly-Arg decreases Thr-Gly-His-Gly-. A 35-residue carboxy-terminal fragment was found to be released.
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PMID:Determination of intermediates, products and cleavage site in the reaction between plasminogen activator inhibitor type-2 and urokinases. 328 Mar 46

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

Plasminogen (PG), urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) are the main molecules involved in fibrinolysis and in many other physiological and pathological processes. In the present study we report that human t-PA, purified from human melanoma cells, and PG, purified from human plasma, both contain P-Tyr residues, as revealed by immunoblotting analyses with monoclonal anti-P-Tyr antibodies. In addition HPLC amino acid analysis of acid-hydrolyzed t-PA, PG and u-PA, shows that: (i) P-Ser and P-Tyr residues are present in t-PA; (ii) P-Thr and P-Tyr are present in PG; (iii) P-Ser, P-Thr and P-Tyr are present in u-PA. The utilization of monoclonal anti-P-Ser and anti-P-Thr antibodies in immunoblotting experiments has confirmed these data which indicate that phosphorylation is a common feature of PAs and of PG.
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PMID:Phosphorylation of human plasminogen activators and plasminogen. 753 24

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-beta 1 on macrophage uPA expression. TGF-beta 1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1 alpha, tumor necrosis factor-alpha, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-beta 1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-beta 1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitor H7 markedly reduced the ability of TGF-beta 1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-beta 1 to upregulate macrophage uPA expression. TGF-beta 1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-beta 1 with either serum or methylamine-modified alpha 2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-beta 1-primed macrophages were cultured on matrices containing bound 125I-bFGF, their release of 125I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-beta to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-beta stimulates angiogenesis.
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PMID:Transforming growth factor-beta 1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor. 768 44

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.
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PMID:Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity. 772 51

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
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PMID:Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. 796 84

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.
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PMID:Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop. 803 24

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.
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PMID:alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells. 811 82

The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC). At least five structurally autonomous regions of this three-domain protein have been found to exist. Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease. The latter undergoes three unfolding transitions in its enzymatically active form. Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps. These are attributed to the denaturation of the two major subdomains of the protease structure. One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C. This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded. The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein. These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins.
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PMID:Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains. 813 Feb 9

The high-affinity receptor that binds human urokinase-type plasminogen activator (hu-PAR) is a glycosyl-phosphatidylinositol (GPI)-anchored cell-surface glycoprotein that belongs to the Ly-6 superfamily of T-cell-activating receptors. Binding of urokinase (u-PA) to u-PAR is species-specific, since neither murine (mu-PAR) nor hu-PAR binds u-PA from the other species. I designed and analyzed a series of exchanges between hu-PAR and mu-PAR in the N-terminal first domain to which ligand-binding function had been independently mapped. Introduction of as few as 13 murine residues (six of 13 variables) into the N-terminal region of hu-PAR abrogated binding to recombinant human pro-u-PA, whereas the opposite chimera, a mu-PAR carrying six of 13 human residues, was positive for binding. Within this region, the mu-PAR domain 1 could be minimally humanized to bind human pro-u-PA by a substitution of as few as four of the six nonconserved residues, thereby identifying the residues arginine-2, lysine-7, threonine-8, and glycine-10 as important in determining binding specificity. By alanine-scanning mutagenesis, a second recognition site within domain 1 was discovered between residues 47 and 53, a segment that is fully conserved between the human and the mouse receptors. Taken together, these results provide identification of two confined subregions within the N-terminal domain of hu-PAR critically involved in pro-u-PA recognition.
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PMID:The N-terminal domain of human urokinase receptor contains two distinct regions critical for ligand recognition. 821 25


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