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: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human plasminogen isolated from the placenta serum fraction by means of affinity chromatography was activated by trypsin being in covalent bond with sepharose. The activation is studied as dependent on pH, temperature and the proenzyme-activator ratio in the presence of 25% glycerol as a stabilizing agent and without it. Utilization of the immobilized trypsin as a plasminogen activator makes it possible to transform completely the proenzyme to plasmin varying the plasminogen-trypsin ratio and time of activation when it is conducted under optimal conditions: in the presence of 25% glycerol at pH 7.0-7.1 and the temperature of 30 degrees C.
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PMID:[Activation of plasminogen with immobilized trypsin]. 3 46

A method for the determination of the fibrinolytic activity of renal tissue obtained by puncture biopsy is suggested. Three cryostate sections are placed on slides, covered by a film of a mixture of bovine fibrinogen and thrombine in saline (in the control section plasminogen activator is pre-destroyed), placed in a thermostate at 37 degrees C for 24 hours, stained with Ehrlich hematoxylin and embedded in glycerol-gelatine. The fibrinolytic activity is evaluated by calculating the ratio expressing the ratio between the lysis surface and the surface of the kidney cortex section. The advantage of the method lies in the technical simplicity combined with the objective evaluation of the results.
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PMID:[Determination of the fibrinolytic activity of renal tissue in children]. 49 83

Human mesangial cells in culture synthesize and secrete plasminogen activator inhibitor 1 (PAI-1) and tissue-type plasminogen activator (t-PA). Phorbol myristate acetate (PMA), a known activator of protein kinase C, induces a three to four-fold increase in t-PA and PAI-1 release over a period of 24 h, whereas cell-associated t-PA and PAI-1 levels remain relatively stable. A similar effect is obtained with oleylacetyl glycerol, a more physiologic protein kinase C activator. The effect of PMA is suppressed in the presence of H7, an inhibitor of cellular protein kinases, and by cycloheximide and actinomycin D, indicating a requirement for de novo protein and RNA synthesis, respectively. Northern blot analysis of PMA-treated cells reveals a rapid and transient increase in PAI-1 mRNA reaching a maximum after 4-8 h, whereas increase in t-PA mRNA levels requires 24 h. Activation of protein kinase A by addition of 8-bromocyclic AMP (8-bromo cAMP) has no significant effect on PAI-1 release but inhibits the PMA-mediated increases in PAI-1 antigen and mRNA. Addition of 8-bromo cAMP alone does not affect t-PA release. When added to PMA-stimulated cells, 8-bromo cAMP inhibits t-PA release in a dose-dependent manner, but causes a superinduction of t-PA mRNA. 8-bromo cAMP also induces a decrease in PMA-stimulated intracellular t-PA release. Similar inhibition is observed after stimulation of endogenous adenylate cyclase with prostaglandin E1 or isoproterenol. This indicates that protein kinase A activation may inhibit PMA-stimulated t-PA release via a post-transcriptional effect, e.g. inhibition of protein synthesis or activation of protein degradation. In conclusion, hormones or mediators which activate protein kinase C can stimulate t-PA and PAI-1 synthesis in human mesangial cells. Protein kinase A activation has no effect on the basal release of PAI-1 and t-PA by human mesangial cells, and, in contrast to endothelial cells, it inhibits both PMA-stimulated PAI-1 and t-PA releases. This cell-specific regulation of t-PA and PAI-1 seems to be mediated by differential transcriptional and post transcriptional mechanisms.
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PMID:Cell-specific regulation of plasminogen activator inhibitor 1 and tissue type plasminogen activator release by human kidney mesangial cells. 155 43

The influence of diacylglycerols, which are physiological activators of protein kinase C, on the production of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) was studied in order to gain insight into the regulation of fibrinolysis by these cells. 1,2-dioctanoyl-sn-glycerol (diC8) stimulated tPA production in a dose- and time-dependent manner. The tPA antigen in cell supernatants increased from 0.9 ng/10(6) cells in unstimulated cells to 12.4 ng (10(6) cells after incubation with 400 microM diC8 for 24 hours. In contrast, PAI-1 production was not influenced by diC8, whereas phorbol 12-myristate 13-acetate (PMA) or thrombin stimulated both, tPA and PAI-1 production by HUVEC. Staurosporine and H7, which are inhibitors of protein kinase C, inhibited tPA synthesis by HUVEC. The degree of inhibition was dependent on the agonist used. While diC8-induced tPA production was inhibited to more than 80% by H7 (10 microM) and staurosporine (10 nM), higher doses of inhibitors were required to inhibit thrombin- and PMA-induced tPA production. Thrombin-induced PAI-1 production was inhibited to more than 80% by H7 (10 microM) and to about 50% by staurosporine, whereas PMA-induced PAI-1 production was not inhibited by staurosporine, and only to about 50% by higher doses of H7 (30 microM). These data suggest that activation of protein kinase C is a common intracellular trigger mechanism for the induction of tPA synthesis by HUVEC. Protein kinase C is most likely also involved in the regulation of PAI-1 synthesis by HUVEC.
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PMID:Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin. 211 75

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.
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PMID:Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C. 271 90

Plasmin is a labile enzyme destroyed by a process termed autodigestion. Studied by a kinetic assay on the substrate Tos-Gly-Pro-Lys-pNA this process is shown to follow a bimolecular mode of reaction, which is retarded by plasmin degradation products. Plasmin is protected by fibrinogen, by epsilon-aminocaproic acid (6-aminohexanoic acid), by increasing ionic strength, and by glycerol. CNBr fragments of fibrinogen did not protect. Lack of substrate protection of plasmin may give rise to errors in a two-stage plasminogen activator assay, while the presence of substrate in a one-stage method prevents degradation of the generated plasmin.
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PMID:The autodigestion of human plasmin follows a bimolecular mode of reaction subject to product inhibition. 293 86

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.
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PMID:Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells. 404 Aug 7

Tissue factor (TF) markedly enhances the ability of factor VIIa (FVIIa) to cleave both macromolecular and small peptidyl substrates. Using soluble mutant TF (sTF) to investigate TF-enhanced FVIIa amidolytic activity in solution, we screened thirty-four commercially available peptidyl-p-nitroanilide substrates and found that substrate hydrolysis rates were influenced by both the peptide sequence and the N-terminal blocking group (MeSO2 > MeO-CO or free N-terminus >> benzoyl). Two substrates (Chromozym t-PA: MeSO2-D-Phe-Gly-Arg-pNA; and CBS 34.47: H-D-cyclohexylglycyl-alpha-aminobutyryl-Arg-pNA) were cleaved at rates higher than those of previously reported chromogenic substrates for FVIIa. The pH range of FVIIa amidolytic activity toward Chromozym t-PA was 6.5 to 10 with an optimum at pH 7.8, while sTF.VIIa had a higher pH optimum (pH 8.4 to 8.5). The degree of enhancement of FVIIa activity by sTF varied from 12-fold at pH 7.5 to 73-fold at pH 9.9. The effect of a variety of agents on FVIIa amidolytic activity was surveyed: most decreased activity, while glycerol and ethylene glycol enhanced the activity of FVIIa but not sTF.VIIa. These results indicate that the effect of sTF on the catalytic center of FVIIa is pH-dependent, and that certain polyalcohols can partially substitute for TF.
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PMID:Importance of substrate composition, pH and other variables on tissue factor enhancement of factor VIIa activity. 816 20

Human cytosolic phospholipase A2 (cPLA2) is an arachidonic acid specific enzyme which may play a role in arachidonic acid release, eicosanoid production, and signal transduction. The PLA2 activity of this enzyme is stimulated by microM levels of Ca2+. Using a pure recombinant enzyme, we have confirmed that cPLA2 is not absolutely dependent on Ca2+, since Sr2+, Ba2+ and Mn2+ also gave full enzyme activity. Heavy metals, in contrast, inhibited enzyme catalysis suggesting the involvement of an essential cysteine residue. In the absence of Ca2+, high salt concentrations overcame the requirement for divalent metals, indicating that Ca2+ is not required for PLA2 catalytic activity. cPLA2 also displays a lysophospholipase (lyso PLA) activity with lysophosphatidylcholine micelles as a substrate. Unlike the PLA2 activity, the lyso PLA activity toward these micelles is not stimulated by Ca2+. However, upon the addition of glycerol or Triton X-100 to the assay, Ca2+ activation is observed, indicating that substrate presentation can affect the apparent Ca2+ dependence. Glycerol was found to be a potent stimulator of lyso PLA activity and specific activities up to 50 mumol min-1 mg-1 were observed. In addition to the PLA2 and lyso PLA activities, we report that cPLA2 displays a relatively low, CoA-independent transacylase activity which produces phosphatidylcholine from lysophosphatidylcholine substrate. The observation of this novel transacylase activity is consistent with the formation of an acyl-enzyme intermediate.
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PMID:Metal ion and salt effects on the phospholipase A2, lysophospholipase, and transacylase activities of human cytosolic phospholipase A2. 848 88

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 medium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solution (4 degrees C) containing [(3)H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis of the ratios of [(3)H] AA and [(14)C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A(2) (PLA(2)) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA(2) activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [micromol phosphorus/L/mg] : 0.197 +/-. 001 at 0 hours) unless energy blockers were used (0.155 +/-.005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/-.001 at 48 hours). This study shows that a net PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA(2) leading to membrane damage.
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PMID:Phospholipid metabolism of hypothermically stored rat hepatocytes. 1053 45


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