Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose.
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PMID:[Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis]. 5 41

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

The present study demonstrates that the fibrinolytic activity is significantly increased and the level of plasminogen antiactivator diminished in the blood of patients with advanced liver cirrhosis and chronic aggressive hepatitis as compared with the values for healthy subjects. Total fibrinogen concentration was similar in patients and controls. However, electrophoresis of plasma with the use of SDS-polyacrylamide gel (3.5%) showed considerable differences in the composition of fibrinogen fractions. Lower molecular weight (LMW and LMW1) clottable protein was significantly (p less than 0.01) increased in the patients. In two out of 22 patients the higher molecular weight (HMW) fraction was virtually absent. In vitro incubation (37 degrees C for 48 hr) of diluted (1:10) plasma from a patient resulted in extensive degradation of a low-solubility fibrinogen fraction (HMW) previously added to the sample. No degradation was observed in any undiluted plasma samples. It is concluded that the increased concentration of lower-molecular-weight forms of clottable protein in the blood of patients with liver disease is probably related to increased in vivo degradation rather than abnormal synthesis. An association rather than a direct correlation with fibrinolytic activity was found.
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PMID:Abnormal fibrinogen heterogeneity and fibrinolytic activity in advanced liver disease. 40 38

Lysosomes (granules) of rabbit PMN leukocytes were extracted with either HCl or H2SO4, and the extracts were chromatographed over Sephadex to separate protein constituents. Some of the low molecular weight cationic proteins homogeneous on SDS PAGE (8% and 12.5% gels) were characterized by electrophoretic mobility in acid gels and by amino acid analysis. A 3,700 dalton polypeptide, rich in arginine and cysteine, prolonged the partial thromboplastin time of normal plasma. In low concentration, this protein shortened the clotting time of pure fibrinogen by thrombin. In high concentration this lysosomal cationic protein precipitated fibrinogen from solution; no fibrinopeptides were released to suggest cleavage of fibrinogen. Fibrinolytic protease activity was detected in crude H2SO4 extracts but not in crude HCl extracts. Two separate plasminogen activators, differing from kallikrein or prekallikrein, were isolated from the H2SO4 lysosomal extract and were partially characterized; neither exhibited proteolytic activity on fibrinogen free of plasminogen.
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PMID:Isolation and characterization of granulocyte lysosomal proteins and study of their effects on the clotting system. 54 40

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

In these studies the fibrinogen plate medium proved to be not sufficiently specific for the demonstration of staphylokinase (SAK). For this purpose we modified the azocasein-test by the addition of plasminogen. Protease, on the other hand, we measured without plasminogen. This enabled us to differentiate between the 2 enzymes. After precipitation with ZnCl2 from the culture supernatant of Staphylococcus aureus strain, V8, SAK could be prepurified by filtration on ultrogel AcA 44 (tab. 1, fig 1). A more than 100-fold increase in specific SAK-activity (in comparison to that in the culture supernatant) to 34.722 units/mg protein was achieved after 2 X isoelectric focusing between pH 3.5-10.0 and refocusing between pH 5.0-7.0 (fig 2). The partially purified SAK was free of protease, coagulase, beta- and delta-hemolysins. DNase, lipase and phosphatases, but it contained minor amounts of alpha-hemolysin. It revealed only 1 band in the SDS-polyacrylamide-gel-electro-phoresis and 1 precipitin-line in the double immunodiffusion test with an antiserum against the SAK preparation after ultrogelfiltration.
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PMID:[Demonstration and partial purification of staphylokinase (author's transl)]. 84 7

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

The specific binding sites for tissue-type plasminogen activator (t-PA) were investigated in human umbilical vein endothelial cells. After adding 125I-t-PA (M.W. 70 kDa) to endothelial cells in suspension culture, the ligand was recovered from the cell extract after disuccinimidyl suberate treatment as a high molecular complex with M.W. of 90 kDa on SDS-PAGE. The complex reacted to only anti-t-PA IgG but not to anti-PAI-1 IgG immunoblot analysis, indicating a t-PA specific binding protein. 125I-t-PA ligand blotting of the cell extract revealed that the binding protein had M.W. 20 kDa. The binding of 125I-t-PA to endothelial cells was reduced in the presence of an excess amount of t-PA, plasminogen and 6-aminohexanoic acid, indicating that the binding sites were also recognized by plasminogen, and that t-PA and plasminogen were bound via lysine binding sites in the molecule. These findings suggest that human endothelial cells have specific t-PA binding molecules which may be expressed on the cell surface as t-PA receptors.
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PMID:Analysis of binding protein for tissue-type plasminogen activator in human endothelial cells. 132 62

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


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