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)

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.
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PMID:Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains. 674 91

The immunochemical properties of high and low molecular forms of urokinase (HMW-UK, MW 53,000, 124,000 IU/mg protein; LMW-UK, MW 32,000, 230,000 IU/mg protein) were studied with specific antisera against the functionally active heavy chain (H chain, MW 31,000, 201,000 IU/mg protein) and the light chain (L chain, MW 18,000) of HMW-UK. Using a double immunodiffusion technique, LMW-UK did not demonstrate L chain antigenicity in the molecule. Anti-L-chain serum exerted no effect on LMW-UK and the H chain, but anti-H-chain serum strongly inhibited the fibrinolytic activity of all the active enzymes (HMW-UK, LMW-UK, and H chain). Anti-L chain serum was found to exert an antifibrinolytic effect on HMW-UK.
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PMID:Immunochemical studies of high and low molecular forms of urokinase. 681 Jun 25

Two forms of urokinase [EC 3.4.99.26] with molecular weights of 51,600 and 34,500 were purified from human urine. The specific activities of the high molecular weight urokinase (HMW-UK) and low molecular weight urokinase (LMW-UK) were 157,400 and 246,700 International Units (IU/mg), respectively. Purified HMW-UK was 97% active and LMW-UK was 88% active, as judged by using p-nitrophenyl-p'-guanidinobenzoate. LMW-UK had five multiple isoelectric subforms, compared with HMW-UK which had only one. Not only HMW-UK but also LMW-UK was composed of two polypeptide chains linked by disulfide bond(s). The molecular weight of the heavy chain of both forms was the same (34,000 daltons), while the molecular weight of the light chain of HMW-UK was 17,600 and that of LMW-UK was approximately 1,200-3,400. Enzyme kinetic studies revealed that the kinetic constants, Km and Kcat, of both forms toward the synthetic substrates, acetyl-Gly-Lys-methylester (AGLMe) and glutaryl-Gly-Arg-4-methylcoumarin-7-amide (GGA-MCA), were almost the same, but the dissociation constant of HMW-UK toward Glu-plasminogen was 2.4-2.6 times less than that of LMW-UK. HMW-UK incubated at 37 degrees C was converted into LMW-UK in an autocatalytic digestion manner leading to no loss of the total activity. These results show that HMW-UK with a higher affinity toward Glu-plasminogen is converted into LMW-UK with a lower affinity, a greater portion of the light chain of HMW-UK splitting off.
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PMID:A comparative study of high molecular weight urokinase and low molecular weight urokinase. 702 50

The single polypeptide chain of native plasminogen (molecular weight approx. 90000) after CNBr-cleavage and gel filtration (Sephadex G-75) yielded a high molecular weight core fraction of fragments linked by disulfide bridges and three fragments of lower molecular weight (N-terminal and C-terminal CNBr-fragments and dodecapeptide). From the reduced and S-carboxamidomethylated core fraction an additional seven fragments with molecular weights between 2000 and 38000 were obtained. The CNBr-fragments were aligned in the porcine plasminogen polypeptide chain according to sequence homologies with the known primary structure of human plasminogen. Due to the lack of two methionine residues in kringle 1 and in the N-terminal part of the light chain region and to an additional methionine residue in kringle 2 the CNBr-fragment pattern differs from that of human plasminogen. Affinity chromatography of elastase-digested, native plasminogen yielded three fragments with intact lysine binding sites, originating from the heavy chain region and a non-adsorbable fragment, corresponding to human 'mini'-plasminogen. This fragment was converted to urokinase into a proteolytically active protein which served for the isolation of the porcine plasmin light chain. With the aid of the fragments produced by the CNBr and elastase cleavage approx. 350 residues were sequenced, of which about 80% showed identity with the sequence of human plasminogen. This percentage varied depending on the region of the molecule, with the highest extent of identity (80--90%) found in the analyzed kringles 2 and 4.
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PMID:Primary structure of porcine plasminogen. Isolation and characterization of CNBr-fragments and their alignment within the polypeptide chain. 734 Dec 39

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.
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PMID:Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein. 749 70

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.
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PMID:Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin. 752 97

The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) is a large cell surface receptor consisting of a 515-kDa heavy chain and an 85-kDa light chain proteolytically derived from a 600-kDa precursor. Previous work has shown that LRP is responsible for mediating the internalization of urinary-type plasminogen activator (uPA) complexed to plasminogen activator inhibitor type I (PAI-1) (Nykjaer et al., 1992; Herz et al., 1992). The current study indicates that pro-urokinase (pro-uPA) and two chain urokinase (tc-uPA) bind directly to purified LRP, and that LRP mediates their internalization and degradation in Hep G2 cells. In vitro binding assays demonstrated that pro-uPA and tc-uPA bind to purified LRP with affinities (Kd = 45 and 60 nM, respectively) that are approximately 15 to 20-fold weaker than the affinity of uPA.PAI-1 complex for LRP (Kd = 3 nM). Competitive binding experiments revealed that pro-uPA and tc-uPA completely inhibit binding of uPA.PAI-1 complexes to purified LRP. The binding of 125I-pro-uPA to LRP is blocked by the 39-kDa receptor-associated protein, but not by an amino-terminal fragment of uPA, which is known to block binding of uPA to the urokinase receptor. 125I-Pro-uPA can be internalized and degraded by Hep G2 cells independent of PAI-1. Both the internalization and degradation are completely blocked by receptor-associated protein or affinity-purified LRP antibodies, indicating that LRP is mediating this process. These processes are also blocked by the amino-terminal fragment, which suggests that the favored pathway for uPA metabolism is initial binding to the urokinase receptor, followed by ligand transfer to LRP, then internalization leading to degradation.
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PMID:Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates cellular uptake of pro-urokinase. 769 18

The human urokinase (uPA) kringle (K) domain has been characterized via high resolution NMR spectroscopy. The 1H spectrum is analogous to that of the K2 domain of tissue-type plasminogen activator (tPA) and other homologous domains from the plasminogen (Pgn) heavy chain. This indicates a similar folding for the uPA/K. Comparisons of the high-field methyl and aromatic regions of the uPA/K and tPA/K2 spectra against those from the Pgn/K1 and K4 homologues afford the immediate assignment of signals stemming from conserved residues, such as the characteristic high-field shifted Leu46 delta, delta'-methyl doublets, and the aromatic side chains at the hydrophobic core, in particular those from Trp25, His48a, Tyr50, and Trp62. Resonances unresolved due to spectral overlaps in the 1H-1H correlated two-dimensional spectra were identified via a natural abundance 1H-13C single/multiple quantum correlated experiment. Spin systems unique to the uPA/K, such as His7, His37, His40, and His78, were assigned from Overhauser experiments and sequence information. Acid/base titrations of His imidazole signals in 2H2O yielded pKa* (pKa determined from acid/base titration in 2H2O, uncorrected for deuterium isotope effects) values of 6.2 for His7, 6.3 for His37, 6.4 for His40, 4.1 for His48a, and 6.2 for His78, which suggests a significant structural protection for His48a, consistent with a buried location within the hydrophobic core. Binding of low molecular weight heparin to the uPA/K in 2H2O affects mainly the His37, His40, His48a, and Tyr50 resonances, in a concerted and saturable fashion (association constant approximately 58 mM-1). The absence of perturbation of the His7 and His78 side chains indicates that segment 37-50 is selectively sensitive to heparin binding. Thus, the kringle outer B-loop is likely to be proximal to the basic residues responsible for the interaction with the polyanion ligand.
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PMID:1H NMR characterization of the urokinase kringle module. Structural, but not functional, relatedness to homologous domains. 831 53

We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).
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PMID:High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells. 844 52

Cleavage of high molecular weight kininogen (HK) by plasma kallikrein results in a light chain and a heavy chain (HK). The light chain has two domains: D6, which binds (pre)kallikrein, and D5, which binds to anionic surfaces, including heparin as well as zinc. Initially, HK was thought to be important for surface-activated coagulation. HKa or D5 binds to the urokinase receptor on endothelial cells, thereby enhancing the conversion of prourokinase to urokinase by kallikrein, and, thus, cell-associated fibrinolysis. HKa or D5 is antiadhesive by competing with vitronectin binding to the urokinase receptor and/or forming a complex with vitronectin. D5 inhibits endothelial cell migration, proliferation, tube formation and angiogenesis, thus modulating inflammation and neovascularization.
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PMID:Role of the light chain of high molecular weight kininogen in adhesion, cell-associated proteolysis and angiogenesis. 1125 75


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