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)

We report a functional assay capable of quantifying prourokinase (ProUK)- like material in plasma where urokinase (UK) is also present. The assay involves inactivation of urokinase with a specific, active site directed irreversible inhibitor, dansyl-glutamyl glycyl arginine chloromethylketone (dansyl- GGACK). Excess inhibitor is subsequently quenched with dithiothreitol (DTT). The ProUK-like material in plasma is then converted to active urokinase with thermolysin, a proteolytic enzyme of bacterial origin. Alpha 2-macroglobulin in plasma inhibits thermolysin; however alpha 2-macroglobulin is inactivated with methylamine. The assay can detect as little as 20 ng of ProUK and is linear from 20 to 120 ng. The assay was applied to quantify the amount of ProUK-like material in plasma obtained from dog at various times after i.v. administration of 100,000 or 75,000 U/kg, of pro-urokinase.
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PMID:A chromogenic enzymatic assay capable of detecting prourokinase-like material in plasma. 163 4

Tumor cell invasion and metastasis is a multifactorial process, which at each step may require the action of proteolytic enzymes such as collagenases, cathepsins, plasmin, or plasminogen activators. An enzymatically inactive proenzyme form of the urokinase-type plasminogen activator (pro-uPA) is secreted by tumor cells which may be converted to an enzymatically active two-chain uPA-molecule (HMW-uPA) by plasmin-like enzymes. Action of proteases on pro-uPA may generate the enzymatically active or inactive high-molecular-weight form of uPA (HMW-uPA). Some proteases (plasmin, cathepsin B and L, kallikrein, trypsin or thermolysin) activate pro-uPA by cleaving the peptide bond Lys158 and IIe159. Other proteases (elastase, thrombin) cleave pro-uPA at different positions to yield enzymatically inactive HMW-uPA. HMW-uPA may be split into the enzymatically active LMW-uPA and the enzymatically inactive ATF (amino terminal fragment). ATF may be cleaved between peptide sequence 20 and 40 within the receptor binding domain of uPA (GFD). Such impaired ATF does not bind to uPA-receptors. Action of the bacterial endoproteinase Asp-N from Pseudomonas fragi mutant on pro-uPA or HMW-uPA, however, generates intact ATF which efficiently competes for binding of HMW-uPA or pro-uPA to receptors on tumor cells. High uPA-antigen content (pro-uPA, HMW-uPA, or LMW-uPA) in breast cancer tissue (not in plasma) indicates an elevated risk for the patient of recurrences and shorter overall survival. Thus pro-uPA/uPA-antigen content in breast cancer tissue serves as an independent prognostic parameter for the outcome of the disease. Cathepsin D is also an independent prognostic factor for recurrences and overall survival. High content of cathepsin D in breast cancer tumors is, however, not correlated with elevated levels of pro-uPA/uPA indicating that synthesis and release of cathepsin D and pro-uPA/uPA are independent events.
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PMID:Biological and clinical relevance of the urokinase-type plasminogen activator (uPA) in breast cancer. 180 51

Human and rabbit kidney and urine contain an inactive form of kallikrein. Studies on the mRNA sequence suggested that the active form of the enzyme and the propeptide are linked by a peptide bond between a basic and hydrophobic amino acid. We studied the activation of prokallikrein by serine proteases and a neutral metalloproteinase, thermolysin, because serine proteases cleave the peptide chain after a basic amino acid and thermolysin before a hydrophobic amino acid. The activity of kallikrein was measured by RIA and with a fluorogenic peptide substrate. Trypsin was used as a standard reference activator. We found that human plasmin and plasminogen, activated by urokinase, activate prokallikrein. Pronase coupled to Sepharose also enhanced the activity of the renal kallikrein zymogen. On a molar basis, thermolysin was a more effective activator of prokallikrein than trypsin. The activation by thermolysin was blocked by the inhibitor phosphoramidon, but not by DFP or SBTI. These experiments indicate that, in addition to serine proteases, neutral metalloproteases of tissues may activate prokallikrein.
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PMID:Activation of human and rabbit prokallikrein by serine and metalloproteases. 315 29

Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M.
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PMID:Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal. 403 Jul 34

A protein capable of inhibiting trypsin and other pancreatic proteases has been purified to homogeneity from Escherichia coli by conventional procedures and affinity chromatography. It is stable for at least 30 min at 100 degrees C and pH 1.0, but it is inactivated by digestion with pepsin. The inhibitor has an apparent molecular weight of 38,000 as determined by gel filtration and must be a homodimer since it contains a single 18,000-dalton subunit upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The inhibitor has an isoelectric point of 6.1. One dimeric molecule of the inhibitor can bind two trypsin molecules to form a mixed tetrameric complex, in which trypsin molecules are completely inhibited. The inhibitor is not digested by the trypsin. When N-benzoyl-DL-arginine-p-nitroanilide was used as a trypsin substrate, half-maximal inhibition was observed at 22 nM. This protein also inhibits chymotrypsin, pancreatic elastase, rat mast cell chymase, and human serosal urokinase, but it does not inhibit human pulmonary tryptase, kallikrein, papain, pepsin, Staphylococcus aureus V8 protease, subtilisin, and thermolysin. Surprisingly, it did not inhibit any of the eight soluble endoproteases recently isolated from E. coli (i.e. proteases Do, Re, Mi, Fa, So, La, Ci, and Pi) nor the chymotrypsin-like (protease I) and trypsin-like (protease II) esterases in E. coli. The inhibitor is localized to the periplasmic space and its level did not change with different growth media or stages of cell growth. The physiological function of this E. coli trypsin inhibitor is unknown. We suggest that E. coli trypsin inhibitor be named "Ecotin."
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PMID:Purification from Escherichia coli of a periplasmic protein that is a potent inhibitor of pancreatic proteases. 641 24

Single-chain urokinase-type plasminogen activator (scu-PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), we found that the lysosomal aminopeptidase dipeptidyl-peptidase I or cathepsin C efficiently activates tcu-PA/T. Cathepsin C was as active towards tcu-PA/T as the bacterial proteinase thermolysin and about 300-times more active than plasmin. The activation by cathepsin C proceeded in a concentration-dependent and time-dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu-PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu-PA/T occurs by cleavage between Lys158-Ile159 and removal of the two N-terminal amino acid residues (Phe157-Lys158) of the B chain of tcu-PA/T. We conclude that both thrombin and dipeptidyl-peptidases like cathepsin C might play a regulatory role in the plasminogen-plasmin system by inactivating scu-PA and activating tcu-PA/T, respectively.
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PMID:Activation of thrombin-inactivated single-chain urokinase-type plasminogen activator by dipeptidyl peptidase I (cathepsin C). 805 19

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 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

The myxoma and malignant rabbit fibroma poxviruses are lethal tumorigenic viruses of rabbits whose virulence is modulated by the production of a virus-encoded secreted serine proteinase inhibitor, SERP-1. This viral protein was detected in medium harvested from myxoma and malignant rabbit fibroma virus-infected cells, and its inhibitory profile has been characterized by gel and kinetic analysis. SERP-1 forms complexes with and inhibits the human fibrinolytic enzymes plasmin, urokinase, and two-chain tissue-type plasminogen activator (association rate constants 3.4 x 10(4), 4.3 x 10(4), and 3.6 x 10(4) M-1 s-1 respectively). It is also able to inhibit C1S, the first enzyme in the complement cascade with an association rate constant which was unaffected by the addition of heparin (1.3 x 10(3) M-1 s-1). SERP-1 acts as a substrate for and is cleaved by thrombin, porcine trypsin, human neutrophil elastase, porcine pancreatic elastase, thermolysin, subtilisin, bovine alpha-chymotrypsin, and factor Xa. Incubation with kallikrein and cathepsin G had no effect. The structure of SERP-1 has been modeled on other members of the serpin family which revealed the characteristic serpin architecture apart from the absence of the D-helix. Structural analysis and kinetic assays demonstrate that the absence of this region does not prevent inhibitory activity and furthermore allow the identification of cysteine residues involved in internal and intermolecular disulfide bonding.
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PMID:Inhibition of plasmin, urokinase, tissue plasminogen activator, and C1S by a myxoma virus serine proteinase inhibitor. 841 56

The bacterial metalloproteinase thermolysin catalyzes the efficient activation of pro-urokinase to an active high-molecular-weight form of the protein. Thermolysin and plasmin convert pro-urokinase to enzymes of essentially equal activities in amidolytic assays, but with different molecular structures. The B-chains of the proteins produced by thermolysin and plasmin are of the same size (33 kDa) and have the same amino-terminal sequences, demonstrating that the cleavage of the Lys158-Ile159 bond of pro-urokinase is catalyzed by both enzymes. However, thermolysin also reacts at additional sites in the growth factor domain of the A-chain at nearly the same rate as that of the activation reaction. Polypeptides derived from hydrolyses of the Glu3-Leu4, Tyr24-Phe25, Asn27-Ile28 and Lys36-Phe37 bonds are recovered after reduction of the activated protein. The carboxy-terminus of the A-chain has been shown to be Arg-156, a consequence of proteolysis of the Arg156-Phe157 bond. In contrast to plasmin, thermolysin activates thrombin-inactivated pro-urokinase nearly as rapidly as it does the native zymogen. Thermolysin provides a useful alternative to plasmin for the catalytic activation and analysis of pro-urokinase, since the bacterial metalloproteinase is stable in solution and not susceptible to inhibition by aprotinin and other serine proteinase inhibitors.
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PMID:Characterization of the activation of pro-urokinase by thermolysin. 842 17


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