scholarly journals Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2283-2290 ◽  
Author(s):  
H Hoogendoorn ◽  
CH Toh ◽  
ME Nesheim ◽  
AR Giles
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Metal Ion ◽  
Protein C ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Molecular Weight Complex

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

scholarly journals Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2283-2290 ◽  
Author(s):  
H Hoogendoorn ◽  
CH Toh ◽  
ME Nesheim ◽  
AR Giles
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Metal Ion ◽  
Protein C ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Molecular Weight Complex

Abstract In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

The polypeptide components of scintillons, the bioluminescence organelles of the dinoflagellate Gonyaulax polyedra

1993 ◽  
Vol 71 (3-4) ◽  
pp. 176-182 ◽  
Author(s):  
Michel Desjardins ◽  
David Morse
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Close Packing ◽  
Gonyaulax Polyedra

Scintillons, the bioluminescence organelles of Gonyaulax polyedra, were purified by isopycnic density gradient centrifugation until only low levels of contaminating chloroplasts and mitochondria were detected by fluorescence and electron microscopy. Purified scintillons catalyzed the luminescent reaction with kinetics identical to those observed during the bioluminescence flash in vivo. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the organelles appeared to contain only two proteins. These proteins were identified as luciferase (135 kilodaltons) and luciferin-binding protein (75 kilodaltons) based on their size and their known functions in the bioluminescence reaction in vitro. The staining of luciferin-binding protein by Coomassie blue was 2.4 ± 0.3 (n = 19) times greater than the luciferase, suggesting that there are four binding protein monomers for every luciferase monomer. A model is proposed for the close packing of the two proteins inside the scintillons.Key words: luciferase, luciferin-binding protein, density gradient centrifugation, dinoflagellate.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals Turnover of *I-protein C inhibitor and *I-alpha 1-antitrypsin and their complexes with activated protein C

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2290-2295 ◽  
Author(s):  
M Laurell ◽  
J Stenflo ◽  
TH Carlson
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Activated Protein C ◽  
Human Protein ◽  
Relative Contribution ◽  
Protein C Inhibitor ◽  
The Difference ◽  
Alpha 1 Antitrypsin

Abstract The rates of clearance and catabolism of human protein C inhibitor (PCI) and human alpha 1-antitrypsin (alpha 1-AT) and their complexes with human activated protein C (APC) were studied in the rabbit. The radioiodinated-free inhibitors had biologic half-lives of 23.4 and 62.1 hours, respectively, while the corresponding *I-labeled activated- protein C complexes were cleared with half-lives of 19.6 +/- 3.1 and 72.2 +/- 6.1 minutes. Complex clearances were linked to their catabolism as shown by a correlation between clearance and the appearance of free radioiodine in the plasma. Thus, the difference in the rates of catabolism would result in a fivefold greater amount of alpha 1-AT-APC complex than PCI-APC complex 1 hour after the formation of equal amounts of these in vivo. These results lead to the conclusion that the relative contribution of PCI and alpha 1-AT to the physiologic inhibition of APC cannot be determined only from the rates of the formation of these complexes in vitro, or from measurement of their levels in plasma. The APC-PCI complex is unstable as compared with the APC-alpha 1-AT complex, compounding the problem of estimating rates of complex formation from their levels in plasma.

scholarly journals Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 722-728 ◽  
Author(s):  
M Geiger ◽  
K Huber ◽  
J Wojta ◽  
L Stingl ◽  
F Espana ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Specific Activity ◽  
Ex Vivo ◽  
Plasminogen Activator Inhibitor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Protein C Inhibitor

Abstract Protein C inhibitor (PCI) and plasminogen activator inhibitor 3 (PAI-3; urinary urokinase inhibitor) are immunologically identical. The role of PCI for urokinase (uPA) inhibition in vivo was investigated. We therefore developed an enzyme-linked immunosorbent assay (ELISA) specific for uPA-PCI complexes: Rabbit anti-PCI IgG was immobilized on a microtiter plate and following incubation with uPA-PCI complex- containing samples, bound uPA-PCI complexes were quantified with a horseradish-peroxidase-linked monoclonal antibody (MoAb) to uPA. Using this assay, time, dose, and heparin-dependent complexes were detected when uPA was incubated with normal plasma or purified urinary PCI, whereas no complexes were measurable using PCI-immunodepleted plasma. Plasma samples (containing 20 mmol/L benzamidine to prevent complex formation ex vivo) from patients undergoing systemic urokinase therapy (1 x 10(6) IU/60 min intravenously [IV]) after myocardial infarction were also studied. uPA present in these plasma samples (up to 1,200 ng/mL) had only 43% to 70% of the specific activity of purified 2-chain uPA, suggesting that a major portion of uPA is complexed to inhibitors. In these plasma samples uPA-PCI complexes were present in a concentration corresponding to 21% to 25% of inactive uPA antigen. These data suggest that at high uPA concentrations, such as during uPA therapy, plasma PCI might contribute significantly to uPA inhibition in vivo.

In Vitro and in Vivo Neutralization of Murine Activated Protein C

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3364-3364
Author(s):  
Laurent Burnier ◽  
Jose A. Fernandez ◽  
John H. Griffin
Keyword(s):  
Active Site ◽  
Protein C ◽  
Activated Protein C ◽  
Activity Level ◽  
Protective Effects ◽  
Wild Type ◽  
Amidolytic Activity ◽  
Endogenous Protein

Abstract Abstract 3364 Activated Protein C (APC) is a circulating serine protease with two major roles to maintain homeostasis. APC acts via multiple receptors, including protease-activated receptor 1, to exert anti-apoptotic and vascular integrity protective effects. A number of protective effects of pharmacologic APC are reported in the literature, with beneficial effects in kidney, brain and irradiation-induced pathologies. The functional protections of the endogenous protein C systems are challenging to study. A better understanding of its mechanisms at different cellular levels and in different tissues is needed to enable evaluation of its further usage in humans. To that end, new tools should be considered to increase our knowledge. To help evaluate the endogenous murine protein C system and to be able to neutralize pharmacologic APC, we have made and characterized a novel rat monoclonal anti-mouse protein C antibody, SPC-54, that almost completely ablates in vitro and in vivo APC activity. In solid phase binding assays, the Kd of SPC-54 for APC was about 8 nM. In biochemical assays, SPC-54 inhibited amidolytic activity of wild-type murine APC by > 95%. SPC-54 was similarly a potent inhibitor (> 90%) of the amidolytic activity of the 5A-APC mutant. IC50 value for wild-type APC and the 5A-APC mutant were comparable. SPC-54 was pre-incubated with APC, followed by the addition of a 20 fold molar excess of biotinylated FPR-chloromethylketone, quantification of biotinylation of APC was readily made by SDS-PAGE and Western blots using infrared-coupled streptavidin. SPC-54 blocked successfully active site titration of APC using this biotinylated active site titrant. These and other experiments suggest that the SPC-54 epitope is located in the vicinity of the active site, such that it blocks different small substrates from reaching the active site. When we performed thrombin generation assays using mouse platelet-poor plasma to check whether SPC-54 was a potent blocker of APC activity in plasma, we showed that SPC-54 neutralized almost completely exogenous APC anticoagulant activity in a dose-dependent manner. Using native polyacrylamide gel migration, Western immunoblotting and immuno-precipitation with protein G-agarose, we confirmed that SPC-54 was bound to protein C in plasma after infusing mice with SPC-54 (10 mg/kg). Moreover, using a modified ELISA that is capable to quantify the pool of activatable protein C, the plasma protein C activity level was considerably decreased (> 80%) in mice after a single injection of SPC-54 (10 mg/kg), and that this effect of neutralizing circulating protein C was sustained for at least 7 days. For in vivo proof of concept, we performed murine tissue factor-induced thromboembolism experiments. Results showed a severe decrease in survival of mice that were pre-infused with SPC-54 when compared to control (survival time of 7 min vs. 42.5 min respectively, P = 0.0016). Moreover, blood perfusion in lungs of mice infused with SPC-54 (10 mg/kg) was dramatically impaired (decrease of 54%, P < 0.0001) as revealed by infrared quantification of Evans Blue dye as marker of vascular perfusion. We also used endotoxemia murine models to assess effects of SPC-54. SPC-54 decreased survival after endotoxin challenge (25 mg/kg, LD50 dose) in mice infused with SPC-54 (10 mg/kg) at 7 hours after LPS. Mortality was 100% after 36 h in the SPC-54 group, whereas controls, which received either boiled SPC-54 antibodies or PBS vehicle, showed a mortality of about 50% (P < 0.001). In summary, SPC-54 is a potent rat monoclonal antibody that neutralizes murine APC activities in vitro and in vivo. Its characteristic ability to dampen the endogenous protein C/APC system is of value to understand better the role of the endogenous protein C system in murine injury models and also to neutralize pharmacologic murine APC. Disclosures: No relevant conflicts of interest to declare.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

scholarly journals Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0143137 ◽  
Author(s):  
Katrin Einfinger ◽  
Sigrun Badrnya ◽  
Margareta Furtmüller ◽  
Daniela Handschuh ◽  
Herbert Lindner ◽  
...  
Keyword(s):  
Protein C ◽  
Binding Protein ◽  
Phospholipid Binding ◽  
Protein C Inhibitor

scholarly journals Characterization, evaluation of nutritional parameters of Radix isatidis protein and its antioxidant activity in D-galactose induced ageing mice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ping Xiao ◽  
Hongzhi Huang ◽  
Xiang Li ◽  
Jianwei Chen ◽  
Jin-ao Duan
Keyword(s):  
Antioxidant Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Optical Microscope ◽  
Human Consumption ◽  
Tissue Samples ◽  
Radix Isatidis ◽  
Sds Page

Abstract Background Radix isatidis (Isatis indigotica Fort.) is an ancient medicinal herb, which has been applied to the prevention and treatment of influenza virus since ancient times. In recent years, the antioxidant activity of Radix isatidis has been widely concerned by researchers. Our previous studies have shown that Radix isatidis protein (RIP) has good antioxidant activity in vitro. In this study, the composition of the protein was characterized and its antioxidant activity in vivo was evaluated. Methods The model of oxidative damage in mice was established by subcutaneous injection of D-galactose for 7 weeks. Commercially available kits were used to determine the content of protein and several oxidation indexes in different tissues of mice. The tissue samples were stained with hematoxylin and eosin (H&E) and the pathological changes were observed by optical microscope. The molecular weight of RIP was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of RIP was determined by a non-derivative method developed by our research group. Results RIP significantly increased the activities of antioxidant enzymes such as SOD, CAT, GSH-Px and total antioxidant capability (TAOC) but decreased the MDA level in the serum, kidney and liver. H&E stained sections of liver and kidney revealed D-galactose could cause serious injury and RIP could substantially attenuate the injury. The analysis of SDS-PAGE showed that four bands with molecular weights of 19.2 kDa, 21.5 kDa, 24.8 kDa and 40.0 kDa were the main protein components of RIP. Conclusions The results suggested that RIP had excellent antioxidant activity, which could be explored as a health-care product to retard aging and a good source of protein nutrition for human consumption.

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