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)

The behaviour of human urokinase and porcine kidney cell plasminogen activator towards some synthetic substrates has been investigated. Although N- benzyloxycarbonylglycylglycyl -L-arginine 4-methyl-7- coumarylamide (Z-Gly-Gly-Arg-Amc) (I), glutaryl-Gly-Arg-Amc (II) and Z-Gly-Gly-Arg-Val-OMe (III) were substrates, Boc-Gly-Gly-Arg-Val-Val-Gly-Gly-OEt (IV) and Z-Ala-Pro-Gly-Arg-Val-Val-Gly-Gly-OEt (V) were neither substrates nor inhibitors. Steady-state kinetic parameters for the hydrolysis of (II) and (III) by urokinase and porcine kidney cell plasminogen activator were similar.
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PMID:The behaviour of urokinase and porcine kidney cell plasminogen activator towards some synthetic peptides. 653 12

Dipeptidyl argininal (arginine aldehyde) affinity resins of general formula R-(X-Y-argininal) (where R = resin matrix and X, Y = amino acids of varied structure) are synthesized in a solid-phase procedure in which the dipeptide (-X-Y-) is first attached to the resin, followed by the joining of the Y amino acid to argininal semicarbazone, and decomposition of the semicarbazone in a methanol/acetic acid/formaldehyde reagent. An R-(Gly-Gly-argininal) resin binds urokinase tightly, but does not bind thrombin. However, thrombin binds strongly to R-(Phe-Pro-argininal), whereas urokinase does not bind. Accordingly, the X-Y-argininal ligands selectively bind proteinases of identical primary binding site specificity to arginine, but different secondary site specificity in -X-Y-. The selectivity is due to an amplification of peptide binding specificity caused by the transition-state analog properties of the ligands. While the affinity constants between peptide aldehyde and proteinase approach those of antibody-antigen interactions, the elution with semicarbazide (aldehyde-trapping reagent) buffers easily remove tightly bound proteinases without proteinase inhibitors or denaturation. Conditions for the binding and elution of proteinases, methods of regeneration and other characteristics of the resins are described.
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PMID:Transition-state affinity chromatography of trypsin-like proteinases with dipeptidyl argininal ligands. 662 59

A plasminogen activator, previously designated as rat urinary esterase A (Nustad, K., and Pierce, J. V. (1974) Biochemistry 13, 2312-2319), was separated from kallikrein of rat urine and purified to homogeneity. In polyacrylamide slab gel electrophoresis, the purified enzyme showed three closely migrating protein bands which were labeled with [14C]diisopropylphosphorofluoridate and stained on a zymogram using the chromogenic substrate methionine-alpha-naphthyl ester. Two chains, heavy chain(s) (Mr approximately 15,800, 14,200) and light chain(s) (Mr approximately 8,850, 8,550), were separated in SDS-polyacrylamide gel under reducing conditions, while two bands (Mr approximately 24,500 and 23,000) were seen under nonreducing conditions. The active site of the enzyme was associated with the heavy chain. The purified enzyme was stained for carbohydrate by the periodic acid-Schiff reagent. Five bands were distinguished in slab gel electrofocusing with isoelectric points ranging from 5.05 to 5.45. The purified enzyme lysed fibrin clots containing plasminogen but not plasminogen-free fibrin. It hydrolyzed benzyloxylcarbonyl-Gly-Gly-Arg-amino-4-trifluoromethyl coumarin, and a Km of 53 microM and a Vmax of 63 mumol/min/mg of enzyme were obtained at pH 8.0 and 37 degrees C. The enzyme cleaved kininogen substrates to produce kinin which was measured by bioassay or radioimmunoassay. The enzyme was inhibited by soybean or lima bean trypsin inhibitor, aprotinin, alpha 1-antitrypsin, phenylmethanesulfonyl fluoride, D-Phe-Phe-ArgCH2Cl, antipain, leupeptin, benzamidine, and pentamidine. Its pH optimum was 8.5 to 9.0; it was unstable on dilution and on heating. On immunoelectrophoresis, an antiserum to the esterase formed precipitin arcs with rat plasma and this enzyme at identical positions, which in turn were different from those formed with kallikrein. This urinary enzyme belongs to the family of serine proteinases and is immunologically related to urinary kallikrein.
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PMID:Purification and characterization of rat urinary esterase A, a plasminogen activator. 668 2

Specific inhibiting IgG antibodies were raised in a rabbit using purified human high molecular weight urokinase as antigen. The antibodies reacted with both high molecular weight and low molecular weight human urokinase using an Ouchterlony double-immunodiffusion technique in such a way that one line of complete identity was obtained. Neither precipitation nor functional inhibition was observed for the tissue-type plasminogen activator. Kinetic studies with plasminogen as a natural high molecular weight substrate or the synthetic low molecular weight p-nitroanilide substrate pyro-Glu-Gly-Arg-pNA revealed, for both substrates, mainly a competitive type of inhibition for the Fab fragments of the specific anti-urokinase antibodies. This characterized anti-urokinase IgG was employed to develop a competitive radioimmunoassay, for human urokinase with 125I-labeled urokinase as tracer. In this radioimmunoassay, the lower detection limit for urokinase antigen was 10 pg/ml sample; the intrassay variation was 2.8%, and the interassay variation was 5.3%. Applying this radioimmunoassay to plasma samples obtained from healthy young volunteers, urokinase antigen could be measured in a concentration of 7.82 +/- 3.97 ng/ml for mean and 6.66 +/- 2.39 ng/ml for women (mean +/- SD).
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PMID:Characterization of a specific anti-human urokinase antibody: development of a sensitive competition radioimmunoassay for urokinase antigen. 671 50

Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (+/- 16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (Mr 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanilide. The fibrinolytic activity as measured by clot lysis in only slightly increased. The physiological significance of the cleavage is discussed.
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PMID:Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin. 689 Dec 67

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 kinetics of urokinase hydrolysis of the low molecular weight substrates--S-2444 (Glu-Gly-Arg-pNA.HCl), N-benzoyl-L-arginine ethyl ester and acetylglycyllysine methyl ester have been studied. The kinetic constants for the hydrolysis reactions have been determined and the appropriate schemes have been proposed. The native enzyme is shown to be highly thermostable.
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PMID:[Kinetics of urokinase hydrolysis of low molecular weight substrates]. 704 94

The activation of plasminogen results from proteolytic cleavage of the Arg560-Val561 bond by plasminogen activators (Sottrup-Jensen et al. PNAS (1975) 72, 2577). This region of the zymogen occurs in a small disulfide loop that must restrict the conformation around this bond. The nonapeptide sequence NH2-Cys-Pro-Gly-Arg-Val-Val-Gly-Gly-Cys-NH2 of plasminogen containing the activator sensitive arginyl valine bond was synthesized by carbodiimide coupling of Boc-Cys-Pro-Gly-OH(S-4-methylbenzyl) to NH2-Arg(NO2)-Val-Val-Gly-Gly-Cys-NH2(S-4-methylbenzyl), followed by HF treatment and K3Fe(CN)6 oxidation to form a disulfide bond. Purified peptide was not a substrate for urokinase (UK) or plasminogen activator (PA) but possessed a slightly inhibitory activity towards PA. Addition of a lysine to the N-terminus of the nonapeptide yielded a decapeptide sequence of plasminogen that was a better substrate for UK but not for PA. The decapeptide inhibits PA slightly but not UK. These results suggest that active site geometry for PA must be more restrictive than that of UK and that other regions may be involved in the productive interactions with the activators inducing a better fit of the cyclic peptide loop.
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PMID:Synthesis and properties of cyclic peptides containing the activation site of plasminogen. 717 5

A fibrinolytic agent purified from the haemolymph, hair secretion and whole body extract of Lonomia achelous (Cramer) cleaves various chromogenic peptide substrates. The best substrate were found to be pyro-Glu-Gly-Arg-pNA (S-2444) followed by D-Pro-Phe-Arg-pNA (S-2302) and Bz-Ile-Glu-(or) Gly-Arg-pNA (S-2222) designed for urokinase, plasma kallikrein and factor Xa, respectively. Using substrate S-2251 we also found a plasminogen activator.
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PMID:Studies of a fibrinolytic enzyme from the larvae of Lonomia achelous (Cramer) using chromogenic peptide substrates. 733 Aug 21

A simple rapid fluorescent method for the detection of plasminogen activator activity of urokinase type (u-PA) in the tear fluid is described. Small filter paper punches were soaked in the substrate solution (Z-Gly-Gly-Arg-trifluoromethylcoumarinyl-7-amide, 1 mg/1 ml) and aprotinin 100 micrograms/1 ml) dissolved in 0.1 M Tris-HCl buffer, pH 7.2 and dried. The dried punches were soaked with tears (by direct contact of the punch with the site where the activity should be assessed or by dropping of 3-5 microliters of tears collected by a glass micropipette). The punches were incubated in a thermostat (37 degrees C) together with punches containing a known u-PA activity (calibrated punches) in preheated (37 degrees C) Petri dishes. In 1 min intervals (during the first 15 min) and in 5 min intervals thereafter the probes were exposed to UV light, and the time of the first appearance of a bright yellow fluorescence was recorded. In punches containing 5 IU u-PA activity fluorescence appeared after 2 min incubation; 2.5 IU were detected after 5 min, 1.25 IU after 15 min, 0.625 IU after 30 min, 0.313 IU after 60 min, 0.156 IU after 90 min, and 0.078 IU after 120 min incubation. This simple method is recommended for use particularly in clinical laboratories. It enables e.g. to obtain a rather quick information about the urokinase activity in the tear fluid and to start the treatment with an appropriate inhibitor, if necessary.
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PMID:Biochemical and histochemical studies of plasminogen activator of urokinase type (u-PA) activity. I. A simple rapid semiquantitative fluorescent method for its detection in the tear fluid. 751 Sep 19


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