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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seven laboratories collaborating in a study of two intermediate purity plasminogen preparations (64/23, 63/6) observed that the amount of activator (urokinase or streptokinase) and the time of activation of plasminogen influenced the amount of plasmin generated. Using casein and a synthetic polypeptide (S-2251) as substrates, the authors subsequently showed that complete activation of plasminogen was difficult to achieve without acitivity losses due to plasmin autodigestion. Comparison of the polypeptide subunits (on SDS electrophoresis) of the various plasminogen activation mixtures with their plasmin activity allowed the conclusion that at maximum generation of plasmin from plasminogen, some plasminogen remains in the form of an inactive plasminogen intermediate (PLG-i).
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PMID:Activation of plasminogen as a feature in its assay. 14 Jan 14

This paper describes an assay for direct measurement of plasminogen activation and its application for determining the kinetic constants and for screening potential inhibitors of the reaction. The assay is based on the conversion of the single chain of 125I-labelled plasminogen to the two chains of 125I-labelled plasmin (EC 3.4.21.7), the latter then being separated from each other and from the plasminogen substrate by electrophoresis under reducing conditions in SDS-polyacrylamide gels. The Km of activator from transformed murine cells for human plasminogen was 180 nM. A broad range of compounds was tested as potential inhibitors of plasminogen activation and of plasmin-catalyzed fibrinolysis respectively, and the two reactions differed qualitatively and quantitatively in their response to previous agents. The principal qualitative difference was in the susceptibility of the reactions to a spectrum of naturally-occurring macromolecular inhibitors: all of the macromolecular inhibitors that blocked the action of plasmin were without effect on murine activator or human urokinase (EC 3.4.99.26). A variety of small molecules inhibited both of the reactions tested, and showed significant quantitative differences; some of these were active at micron concentrations. The exacting specificity of plasminogen activators for macromolecules, both substrates and inhibitors, encourages the expectation that effective inhibitors of great specificity may be isolated from as yet undiscovered natural sources.
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PMID:Plasminogen activator from cells transformed by an oncogenic virus: inhibitors of the activation reaction. 21 32

Serine proteases or esterases released from cell cultures into the growth medium were converted to radioactive derivatives by active site labeling with tritiated DFP, both in the presence and absence of other competing active site reagents. The individual labeled enzymes were then identified by SDS-polyacrylamide gel electrophoresis and scintillation autoradiography. Conditioned medium from embryonal mouse fibroblasts transformed by mouse sarcoma virus contained five serine enzymes that were not present in medium from normal cells; two serine enzymes were released by both cell types, and one serine enzyme was found only in medium from normal cells. Two of the enzymes released by transformed cells were identified as plasminogen activators; these accounted for most of the serine enzyme labeling in transformed culture media and for most of the serine enzyme difference between normal and transformed cultures. The culture fluids from two cell strains of human neoplastic origin were examined by the same method. A rhabdomyosarcoma strain released eight serine enzymes (mol wt ranging from 22,500 to 102,000), four of which were plasminogen activators; seven serine enzymes (mol wt 26,000-102,000), including two plasminogen activators, were detected in medium from human melanoma cultures. In terms of electrophoretic mobility two of the plasminogen activators from rhabdomyosarcoma were identical with those from melanoma cultures, while the remaining two rhabdomyosarcoma activators coincided with activators found in commerical urokinase.
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PMID:Serine enzymes released by cultured neoplastic cells. 63 49

D was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl esters (BAME) was the most sensitive substrate for D among those tested. The substrate profile of D was dinstinct when compared to that of CIs, CIr, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.
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PMID:Human factor D of the alternative complement pathway: purification and characterization. 87 24

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.
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PMID:Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor. 128 93

Two types of plasminogen activator (PAs) are present in human endometrium, and their contents vary with the different phases of menstrual cycle, i.e. high in the proliferative phase and low in the secretory phase. In the present study by immunohistochemical technique, both uPA and tPA antigens were demonstrated in the stromal and glandular cells of the endometrium. In cell culture, tPA was released only from stromal cells and uPA only from glandular cells as determined by SDS-PAGE followed by fibrin overlay technique, but PA inhibitor type-1 (PAI-1) was secreted by both stromal and glandular cells. Furthermore, secretion of PAs from endometrial cells was enhanced by adding estradiol and markedly inhibited by progesterone in a dose dependent manner, while the PAI reacted just in the opposite way. The effect of the peptide hormones, hCG, GnRH, PRL, as well as cAMP in cell culture on the secretion of PAs and PAI was similar to that of estradiol, while forskolin demonstrated definitely more stimulative effect on tPA than uPA. Taking into account of the finding of the present study, it appears that, under hormonal control, a balance between PAs and PAI in the endometrium exists. The physiological roles of the PAs and PAI in the endometrium were discussed.
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PMID:[Plasminogen activators and plasminogen activator inhibitor type-1 in human endometrium]. 129 66

A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.
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PMID:Characterization of a recombinant chimeric plasminogen activator composed of a fibrin fragment-D-dimer-specific humanized monoclonal antibody and a truncated single-chain urokinase. 131 61

The cellular urokinase-type plasminogen-activator (uPA) receptor (uPAR) is a glycolipid-anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand-binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol-specific phospholipase C (PLC) or endoproteinase Asp-N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem-cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA-binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH-affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR-specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns-specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C-terminus close to the proposed glycolipid-attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as-yet-unidentified transamidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.
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PMID:A soluble form of the glycolipid-anchored receptor for urokinase-type plasminogen activator is secreted from peripheral blood leukocytes from patients with paroxysmal nocturnal hemoglobinuria. 132 6

Two plasminogen activators (PAs): tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as the type-1 plasminogen activator inhibitor (PAI-1) are synthesized and secreted by rat astrocytes. Preliminary studies suggest that PA activity plays a role in astrocyte development and differentiation. We have examined the regulation of the PA system by the cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in purified rat astrocyte cultures. PKA activity was increased by exposing cultured astrocytes to forskolin or dibutyryl cyclic AMP, whereas PKC activity was stimulated with phorbol-12-myristate 13-acetate (PMA). Activation of both second-messenger pathways produced a time- and dose-dependent increase in the total PA activity. However, based on SDS-PAGE/zymography we found that forskolin increased t-PA activity and reduced u-PA activity, whereas PMA treatment caused a significant increase in u-PA activity without altering t-PA activity. Reverse zymography analysis revealed that astrocyte PAI-1 activity is decreased by forskolin and increased by PMA. Together, these results demonstrate that the components of the PA system in rat astrocytes are independently and reciprocally regulated by PKA and PKC. Our findings raise the possibility that the plasminogen activator system could be involved in some of the actions of growth factors and/or neuromodulators that modulate PKC or PKA in astrocytes.
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PMID:Regulation of plasminogen activators and type-1 plasminogen activator inhibitor by cyclic AMP and phorbol ester in rat astrocytes. 133 67

The anti-urokinase-IgG-resistant plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells (ATCC, CCL186) was purified to homogeneity from serum-free conditioned medium by a four-step procedure. The fibroblast plasminogen activator was identified as tissue plasminogen activator (t-PA) by the N-terminal sequence of the purified material and the complete amino acid sequence deduced from its complementary DNA (cDNA). The apparent molecular weight was the range of 64,000 to 68,000 by SDS-PAGE and was in the range of 69,000 to 72,000 by gel filtration. The fibroblast t-PA showed a stricter substrate specificity than urokinase in enzymatic hydrolysis of various chromogenic substrates. Compared to urokinase, the fibrobrast t-PA was more stable by heating at 95 degrees C for five min and was stable from pH 5 to 10. The fibrorast t-PA had a higher affinity for fibrin than urokinase.
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PMID:Purification and characterization of tissue plasminogen activator secreted by human embryonic lung diploid fibroblasts, IMR-90 cells. 136 81


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