Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
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PMID:The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein. 1 96

The study of the interaction between activated protein C (APC) and non-plasmatic inhibitors allowed us to demonstrate that aprotinin is a potent competitive inhibitor of APC with a Ki of 1.35 mumol/L. It was possible to adsorb immunopurified protein C (PC) activated by venom activator to insolubilized aprotinin and to recover the active enzyme after elution by HCl 0.1 N or by a chaotropic ion, for example KSCN 3 mol/L. The interaction involved the active-site of the enzyme since PC and DIP-APC did not bind to the matrix. Thus, APC could be purified, after activation, in a one-stage procedure out of a mixture of protein such as a prothrombin complex concentrate.
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PMID:Inhibition of activated protein C by aprotinin and the use of the insolubilized inhibitor for its purification. 169 90

This report documents attempts to mimic the rate enhancement effect of thrombomodulin on human alpha-thrombin-catalyzed activation of human protein C in the absence of exogenous calcium. Specifically the following tryptamine analogs at 1 mM concentration were shown to enhance the protein C activation rate relative to a control with no added effector at pH 8.3 (50 mM Tris-HCl, 0.1 M NaCl, 37 degrees C): serotonin, 1.2; tryptamine, 2.9; 5-fluorotryptamine, 4.4; 6-fluorotryptamine, 7.2. At much higher levels, e.g. 10 mM, all of the above effectors, as well as indole, showed a moderate inhibition of human protein C activation. ATP, a platelet release product, showed a sigmoidal inhibition pattern similar to that found previously for thrombin amidase, clotting, and esterase activity (Conery, B.G., and Berliner, L.J. (1983) Biochemistry 22, 369-375). Overall, the enhancement factors for human alpha-thrombin activation of protein C with the tryptamine analogs described above were remarkable when considering the effect of a simple ligand versus the natural activator, thrombomodulin.
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PMID:Ligands which effect human protein C activation by thrombin. 365 43

A method for the quantitation of beta-hydroxyaspartic acid in proteins is described. After hydrolysis in 6 M HCl, the beta-hydroxyaspartic acid released is quantitated on an automatic amino acid analyzer employing a pH 2.0 eluting buffer and postcolumn reaction with o-phthalaldehyde for detection. The sensitivity is about 0.01 nmol. Among vitamin K-dependent proteins, factor IX, factor X, protein C, and protein Z each contain about one residue of beta-hydroxyaspartic acid whereas protein S contains two or three residues. Prothrombin lacks beta-hydroxyaspartic acid as do a number of non-vitamin K-dependent proteins also analyzed.
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PMID:Beta-hydroxyaspartic acid in vitamin K-dependent proteins. 663 Jan 96

The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).
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PMID:Role of activated protein C in facilitating basement membrane invasion by tumor cells. 826 50

Human recombinant prethrombin-2 was produced in Escherichia coli. The expressed prethrombin-2 formed intracellular inclusion bodies from which the protein was refolded by a simple one-step dilution process in buffer consisting of 50 mM Tris-HCl, containing 20 mM CaCl(2), 500 mM NaCl, 1 mM EDTA, 600 mM arginine, 1 mM cysteine, 0.1 mM cystine, 10% (v/v) glycerol, and 0.2% (w/v) Brij-58 at pH 8.5. After refolding, prethrombin-2 was purified by hirudin-based COOH-terminal peptide affinity chromatography, and then activated with Echis carinatus snake venom prothrombin activator (ecarin). The activated protein, alpha-thrombin, was then tested for several activities including activity toward chromogenic substrate, release of fibrinopeptide A from fibrinogen, activation of protein C, and thrombin-activatable fibrinolysis inhibitor, reactivity with antithrombin, clotting activity, and platelet aggregation. The kinetic data showed no differences in activity between our recombinant alpha-thrombin and plasma-derived alpha-thrombin. The yield of refolded recombinant human prethrombin-2 was about 4-7% of the starting amount of solubilized protein. In addition, the final yield of purified refolded protein was 0.5-1%, and about 1 mg of recombinant prethrombin-2 could be isolated from 1 liter of E. coli cell culture.
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PMID:An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant-derived alpha-thrombin. 1148 Oct 45

Pseudomonas aeruginosa PAO1 was used in this study. Isolation of outer membrane was accomplished by treating the cell envelope with EDTA and lysozyme, followed by centrifugation. The outer membrane (10 mg of protein) was mixed with 34 mmol/L octyl beta-glucoside-5 mmol/L EDTA-10 mmol/L Tris-HCl (pH 8.0) and subjected to supersonic oscillation for 2 min. The centrifuged supernatant (100 kgf for 30 min at 20 degrees C) was applied onto a DEAE ion-exchange high performance liquid chromatographic column (TSK gel-DEAE-5PW column, 0.75 cm x 7.5 cm i.d.) that was equilibrated with a solution of 10 mmol/L Tris-HCl buffer (pH 8.0) containing 2.5 mmol/L beta-C12E8 and 1 mmol/L EDTA. The column was washed with the same solution and eluted with a linear gradient of 0-0.5 mol/L NaCl in the same solution and fractions A, B, C were collected. Proteins in these fractions were analyzed by SDS-polyacrylamide gel electrophoresis and quantified by the method of Lowry et al. Protein E(Mr 43,000), G(Mr 25,000) and H (Mr 19,000) flowed through the column without adsorption in fraction A. Protein C(Mr 70,000), D(Mr 46,000) and a small amount of F (Mr 34,000) were eluted in fraction B. Fraction A was concentrated with ultrafiltration and applied again onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L beta-C12E8 and 1 mmol/L MgCl2. Fraction B was subjected to DEAE ion-exchange HPLC column in the presence of EDTA. This fraction was then applied onto a DEAE ion-exchange HPLC column equilibrated with 10 mmol/L Tris-HCl buffer, pH 8.0, containing 34 mmol/L octylglucoside and 1 mmol/L EDTA. By these procedures protein C, D and E were purified to apparent homogeneity as judged by SDS-PAGE. In this work, we purified the outer membrane proteins of Pseudomonas aeruginosa, and used a new technique selectively solubilizing the cytoplasmic membrane with sodium lauryl sarcosinate for isolating the outer membrane proteins of Pseudomonas aeruginosa because of its relative simplicity.
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PMID:[Purification of outer membrane proteins in Pseudomonas aeruginosa by high performance ion-exchange liquid chromatography]. 1254 27

Acid aspiration causes direct lung damage and secondary inflammatory response involving several cytokines and accumulation of neutrophils. Activated protein C (APC) exhibits antithrombotic and anti-inflammatory properties. We examined the effect and mechanism of pre-treatment APC on acid-aspirated lung injury in rats. Anesthetized rats were instilled intratracheally with normal saline (NS, 2 ml kg(-1)) or hydrochloric acid (HCl, 0.1 N, 2 ml kg(-1)). Thirty minutes before HCl instillation, APC (200 U kg(-1) h(-1)) was infused continuously into the right jugular vein. Animals were ventilated during the experiments. Five hours after HCl or NS instillation, bronchoalveolar lavage fluid (BALF) and lung tissue samples were obtained. Total and differential cell count, absorbance, albumin concentration, concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC) in BALF, wet and dry weight (W/D) ratio were measured. Platelet count and fibrin degradation product (FDP) in peripheral blood were also measured. HCl instillation markedly increased these values in BALF as well as W/D ratio. APC attenuated the parameters increased by HCl-induced lung injury in rats. However, HCl instillation and APC treatment did not cause significant changes in platelet count and FDP compared with the control. We conclude that APC treatment protected the rats against HCl-induced lung injury and that this action seemed to be due to the anti-inflammatory properties of this protein rather than its anti-coagulant effects.
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PMID:Activated protein C attenuates acid-aspiration lung injury in rats. 1577 12

The flue gas cleaning system of a MSW incinerator with a capacity of 350 kt/year was changed to improve the HCl elimination efficiency. Instead of the semi-wet operating spray reactor and subsequent baghouse, a two-step wet flue gas cleaning was added behind the baghouse. Elemental composition, X-ray powder diffraction patterns and TGA measurements showed that the resulting APC residue was totally different from the former residue. As a consequence, leaching characteristics of both residues also differed and another treatment was required prior to disposal. For the former residue, mainly leaching of Pb (>100 mg/l), necessitated treatment prior to landfilling. The lower alkalinity of the new residue resulted in a leachate pH of 9.7 and a Pb concentration of 0.8 mg/l. The leachate pH of the former residue was 12.4. The leaching of Pb and Zn increased above 100 mg/l when immobilising the new residue with cement. Better results were obtained when immobilising with micro silica. The high CaCl2 x 2H2O content of the new residue brought along clogging of the bag filter system. Adding 1.4% of CaO (or 1.9% of Ca(OH)2) to the residue already improved these inconveniences but again significantly changed the leaching behaviour of the residue.
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PMID:Effect of improving flue gas cleaning on characteristics and immobilisation of APC residues from MSW incineration. 1638 67

Affinity purification of proteins using antibodies coupled to beads and subsequent mass spectrometric analysis has become a standard technique for the identification of protein complexes. With the recent transfer of the isotope dilution mass spectrometry principle (IDMS) to the field of proteomics, quantitative analyses-such as the stoichiometry determination of protein complexes-have become achievable. Traditionally proteins were eluted from antibody-conjugated beads using glycine at low pH or using diluted acids such as HCl, TFA, or FA, but elution was often found to be incomplete. Using the cohesin complex and the anaphase promoting complex/cyclosome (APC/C) as examples, we show that a short 15-60 min predigestion with a protease such as LysC (modified on-bead digest termed protease elution) increases the elution efficiency 2- to 3-fold compared to standard acid elution protocols. While longer incubation periods-as performed in standard on-bead digestion-led to partial proteolysis of the cross-linked antibodies, no or only insignificant cleavage was observed after 15-60 min protease mediated elution. Using the protease elution method, we successfully determined the stoichiometry of the cohesin complex by absolute quantification of the four core subunits using LC-SRM analysis and 19 reference peptides generated with the EtEP strategy. Protease elution was 3-fold more efficient compared to HCl elution, but measurements using both elution techniques are in agreement with a 1:1:1:1 stoichiometry. Furthermore, using isoform specific reference peptides, we determined the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. In summary, we show that the protease elution protocol increases the recovery from affinity beads and is compatible with quantitative measurements such as the stoichiometry determination of protein complexes.
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PMID:Lesson from the stoichiometry determination of the cohesin complex: a short protease mediated elution increases the recovery from cross-linked antibody-conjugated beads. 2104 28


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