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 stabilities of urokinase (UK) in aqueous solution were investigated at pH 5.0-8.0 in the presence (1.0-3.0 x 10(-3) M) and absence of sodium bisulfite (SBS) both under scattered light (1000 lux) and in the dark using the fluorogenic substrate method. Increasing concentrations of SBS tended to increase the inactivation of UK. In the presence of SBS, with the increase in the pH value, UK gained in stability in the pH range of 5.0-8.0. The stability of UK in the presence of SBS in the dark was larger than that under scattered light, especially at pH 5.0. Therefore, it was suggested that the difference in the residual activities of UK between under light and in the dark was due to free radicals formed during the autooxidation of bisulfite under scattered light. UK was stabilized by glucose in the presence of SBS both under scattered light and in the dark. One reason for this phenomenon was postulated to be the formation of inactive bisulfite-glucose addition compound. The degradation products of UK during storage in a solution containing SBS were investigated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. UK was revealed to be split into M.W. 36000 form and M.W. 20000 form by SBS.
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PMID:Stability of urokinase in solutions containing sodium bisulfite. 220 81

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.
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PMID:Synthesis of urokinase-type plasminogen activator and of type-1 plasminogen activator inhibitor in neuronal cultures of human fetal brain: stimulation by phorbol ester. 221 17

Vein graft failure remains a major challenge for the vascular surgeon. Thrombolysis of occluded vein grafts has shown promising short-term results in restoring vein graft patency, however, the long-term results are not established. This study examines the long-term patency and limb salvage after successful thrombolysis and revision of 22 thrombosed vein grafts in 21 patients. There were 17 men and four women with an average age of 60 years (38 to 77 years). Failed vein grafts had an average primary patency of 19 months (1 to 84 months) and included eight in situ grafts and 14 non-in situ grafts. Twelve grafts were to the popliteal level, whereas 10 were infrapopliteal. Thrombolytic agents used included urokinase (15), tissue plasminogen activator (5), and streptokinase (2). After successful thrombolysis, 19 grafts underwent 26 additional procedures including percutaneous transluminal angioplasty (9), vein patch angioplasty (4), vein interposition or jump extension graft (9), or other procedures (4). Three patients had no additional procedure, but one was placed on sodium warfarin (Coumadin). After successful initial vein graft salvage, life-table analysis revealed a 36.6% +/- 11.9% patency at 1 year and a 22.9% +/- 11.6% patency at 3 years. After secondary failure six patients had further interventions contributing to an improved limb salvage of 66.9% +/- 11.6% at 1 year and 60.3% +/- 19.0% at 3 years. The results suggest that thrombosed vein grafts initially salvaged with thrombolysis and revision do not have a favorable long-term patency, and that a premium must be placed on the detection of the failing vein graft before thrombosis.
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PMID:Observations on the use of thrombolytic agents for thrombotic occlusion of infrainguinal vein grafts. 229 48

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.
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PMID:Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin. 241 80

We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.
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PMID:Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells. 241 59

This paper reviews the properties and regulation of single amiloride-sensitive Na+ channels in the apical membrane, and Cl- and K+ channels in the basolateral membrane of rabbit urinary bladder. According to fluctuation analysis, there is an average of one amiloride-sensitive Na+ channel for every 40 micron2 of apical membrane. Each Na+ channel passes 0.7 pA of current under normal, short-circuit conditions. Apical channels are hydrolysed by the endogenous enzyme urokinase, which is released into the urine by the kidney. After exposure to urokinase, the Na+ channel loses its amiloride sensitivity, and eventually becomes unstable in the membrane. The selectivity and kinetic properties of single anion and K+ channels in the basolateral membrane were also studied using the patch clamp technique. The properties of these channels are discussed in terms of the regulation of transepithelial Na+ transport.
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PMID:Apical and basolateral membrane ionic channels in rabbit urinary bladder epithelium. 241 8

Activity of peritoneal plasminogen activator and its regulation by dextran and other macromolecules that clinically suppress postoperative adhesions was studied. Plasminogen activator activity was assayed by a two-stage globinolytic assay that monitors formation of plasmin, as well as by cleavage of a chromogenic peptide substrate (S-2444) in the presence of aprotinin (Trasylol). Plasminogen activator activity was located on the outer surface of human peritoneum. Incubation of peritoneal tissue with buffer in vitro (conditioning) prompted release of plasminogen activator into the conditioning medium. The released plasminogen activator formed a single band on sodium dodecyl sulfate-gel electrophoresis at an apparent molecular weight of 174,000 and was markedly suppressed by antiserum raised against human melanoma tissue-type plasminogen activator. Nonspecific proteolytic activity did not accumulate in the medium during conditioning. The presence of dextran 80 during conditioning of peritoneum reversibly suppressed tissue-bound plasminogen activator activity and reduced plasminogen activator activity in the spent medium. A similar inhibition of peritoneal plasminogen activator was induced by dextran 500, methyl cellulose, and polyvinylpyrrolidone. Dextran, when added to the medium after conditioning, had no direct inhibitory effect on plasminogen activator activity. Dextran did not induce peritoneal production of inhibitor(s) of trypsin, chymotrypsin, or urokinase. On the basis of these findings, two possible mechanisms for the effect of viscous polymers in the reduction of adhesion formation are proposed. These mechanisms consider the importance of peritoneal tissue-type plasminogen activator for removal of fibrin clots and suggest that polymer coating either prevents the shedding of plasminogen activator into the abdominal cavity or reduces the access of fibrin clots to the serosal surfaces.
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PMID:Effect of viscous macromolecules on peritoneal plasminogen activator activity: a potential mechanism for their ability to reduce postoperative adhesion formation. 245 68

The activity of intact urokinase (UK) and urokinases modified by soluble macromolecules (dextran and dextran sulfate sodium) in a mouse body was traced after injection of the 125I-labelled enzymes. The residual fraction of the enzyme in blood can be correlated with the time (t) as follows; Xb = Ae-at + Be-bt + Ce-ct Since a greater than b greater than c, the residual fraction of the enzyme in blood chiefly depends on the magnitude of parameter C. The constant C for modified urokinase was larger than that for urokinase, showing the relative residual in blood was increased by modification of the enzyme. The apparent utilization in 50 min was 13.8% for intact UK, 28.1% for the dextran-UK and 25.2% for the sulfate dextran-UK. Therefore, the apparent utilization of UK in blood was approximately doubled by the modification. Since the half-lives of UK and modified UK in kidney and liver were not long, there were no unacceptable accumulation of the enzymes.
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PMID:Distribution model for the intact urokinase and urokinases modified by soluble macromolecules in rat and mouse bodies. 246 42

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.
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PMID:Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin. 247 Jul 35

The human breast cancer cell line MCF-7 produces a number of estrogen-regulated proteins, among which is tissue plasminogen activator (tPA). Increased medium concentrations of PA activity were observed after the addition of 17 beta-estradiol to cultures of MCF-7 cells. However, in the current study these hormone-regulated increases are limited to cultures near or at confluence, but not in the preconfluent period. MCF-7 cell cultures produce either tPA activity alone or in combination with urokinase activators. At confluence, a single exposure to 17 beta-estradiol stimulates a marked transitory rise in tPA activity in the extracellular and cell-associated compartments; the peak increases were at 48 h for medium activity and 24-48 h for cell-associated activity. Sustained exposure to hormone leads to a persistent increase in activity in both compartments. Examination of the structure-function relationships of estrogen agonists, steroidal and nonsteroidal, as well as nonestrogenic steroids indicated that stimulation of PA activity was restricted to estrogen agonists. The increased activity was reflected in enhancement of tissue PA activity when viewed using sodium dodecyl sulfate-polyacrylamide gel zymography. Those cultures expressing both activators revealed no alteration of urokinase activity due to hormone addition. Antiestrogens added to MCF-7 cells not rigorously limited in exogenous estrogens selectively suppressed tissue PA activity, but not that of urokinase. These data indicate that at the point when MCF-7 cell cultures are no longer growing exponentially, addition of estrogen agonists at physiological concentrations elevates tPA activity while not altering expression of urokinase activity. The discussion suggests a possible role that this regulation may subserve in the function of breast epithelial cells.
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PMID:Estrogen regulation of human breast cancer cell line MCF-7 tissue plasminogen activator. 250 Mar 32


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