Homologues Of Fibrin X Oligomers

1981 ◽  
Author(s):  
R Hafter ◽  
H Graeff ◽  
R v Hugo
Keyword(s):  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Simultaneous Action ◽  
Coagulation Disorder ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Sds Page ◽  
D Dimer

Crosslinked fibrin derivatives signalize intravascular coagulation. D-dimer, Y-D and X oligomers are observed in plasma from obstetric patients with severe coagulation disorder. They are also found in ascitic fluid from patients with advanced ovarian cancer and can be produced in vitro by simultaneous action of thrombin, plasmin and factor XIII with fibrinogen. The study was aimed to evaluate the subunit structure of separated molecular entities. The derivatives were separated by 4% SDS-PAGE preceded in case of the in vivo products by gel filtration and/or by immunoabsorption technique. The gels were sliced at the respective migration positions and derivatives therein reelectrophoresed on 7,5% gels after reduction. Subunit characterisation revealed that D-dimer is composed of the chain remnants γ1-γ1, β2, α2, while Y-D is composed of γ-γ1, β2, α3, α2, besides αE, βE and γE Crosslinked X oligomers are composed of γ-γ, γ-γ1, β, β1, γ2, α1 and α2 besides αE, βE and γE Three possible combinations of plasmin degraded and undegraded dimeric γ-chains were observed in vivo and in vitro: γ-γ γ-γ1 and γ1-γ1. The ratio of degraded (γ1) to undegraded γ-chains in dimeric γ-chain patterns indicates the mol. structure of the respective derivative. Two X oligomers could be demonstrated in which the ratio of γ-γ to γ-γ1 in terms of stain intensity was either 1:1 or 2:1. Their subunit compositions are in accordance with structures describ- able as D-X-Y and D-X-X-Y. Their molecular weights, calculated from the subunit compositions are 476,000 and 716,000 respectively. - It is proposed that crosslinked X oligomers exist as a homologous family with increasing X fragment content.

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 Calcium-binding calmyrin forms stable covalent dimers in vitro, but in vivo is found in monomeric form.

2005 ◽  
Vol 52 (2) ◽  
pp. 469-476 ◽  
Author(s):  
Adam Sobczak ◽  
Magdalena Blazejczyk ◽  
Grzegorz Piszczek ◽  
Gang Zhao ◽  
Jacek Kuznicki ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Binding ◽  
Protein Concentration ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Biologically Active ◽  
Cell Extracts ◽  
Sds Page

The EF-hand Ca(2+)-binding protein calmyrin is expressed in many tissues and can interact with multiple effector proteins, probably as a sensor transferring Ca(2+) signals. As oligomerization may represent one of Ca(2+)-signal transduction mechanisms, we characterised recombinant calmyrin forms using non-reducing SDS/PAGE, analytical ultracentrifugation and gel filtration. We also aimed at identification of biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(2+)-bound calmyrin oligomerizes forming stable intermolecular disulfide bridges. Ultracentrifugation indicated that at a 220 microM initial protein concentration apo-calmyrin existed in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer (trimeric or tetrameric species were not detected). The dimerization constant was calculated as Ka = 1.78 x 103 M(-1) at 6 degrees C. Gel filtration of apo- and Ca(2+)-bound calmyrin at a 100 microM protein concentration confirmed an equilibrium of a monomer and a covalent dimer state. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(2+). However, when calmyrin forms were analyzed under non-reducing conditions in cell extracts by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brain. Moreover, in contrast to recombinant calmyrin, crosslinking did not preserve any dimeric species of calmyrin regardless of Ca(2+) concentrations. In summary, our data indicate that although calmyrin forms stable covalent dimers in vitro, it most probably functions as a monomer in vivo.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

scholarly journals The purification and properties of Factor X from pig serum and its role in hypercoagulability in vivo

1973 ◽  
Vol 133 (2) ◽  
pp. 311-321 ◽  
Author(s):  
R. J. Dupe ◽  
R. M. Howell
Keyword(s):  
Molecular Weight ◽  
Thrombus Formation ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor X ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Initial Adsorption

The molecular weights or shapes of Factor X preparations determined by gel filtration were dependent on the density of the BaSO4 used for the initial adsorption from serum. One form obtained with BaSO4 of density 2g/ml behaved as if it had a molecular weight of 39000 and possessed preformed clotting activity (Factor Xa), whereas that of the form adsorbed with BaSO4 of density 1g/ml had a molecular weight of 69000 and consisted of inactive Factor X precursor. Thus degradation accompanied by activation seems to occur as a result of surface adsorption on high-density BaSO4 and is associated with an interchange of protein between the two bands observed electrophoretically. The clotting and esterase activities measurable in vitro after complete activation were not matched by a corresponding ability to induce thrombus formation and ‘lethality’ in vivo. The most effective preparations of Factor X in this respect possessed preformed activity, which was enhanced in the presence of phospholipid. Factor X lost activity more rapidly in dilute solution, and its concentration at the surface of phospholipid micelles probably decreases loss by dilution in circulating blood.

scholarly journals Isolation and partial chemical characterization of macrophage-derived neutrophil chemotactic factor

1995 ◽  
Vol 4 (4) ◽  
pp. 257-262 ◽  
Author(s):  
M. Dias-Baruffi ◽  
M. C. Roque-Barreira ◽  
F. Q. Cunha ◽  
S. H. Ferreira
Keyword(s):  
Neutrophil Migration ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Chemotactic Factor ◽  
Sds Page ◽  
Neutrophil Chemotactic Factor ◽  
Partial Chemical

Macrophages stimulated with lipopolysaccharide (LPS) release a factor (MNCF; macrophage-derived neutrophil chemotactic factor) which induces neutrophil migrationin vivoandin vitro. Thein vivochemotactic activity of crude MNCF is not affected by pretreating the animals with dexamethasone, an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. We purified MNCF by affinity chromatography of the supernatant from LPS-stimulated macrophages on immobilized D-galactose, followed by gel filtration of the sugar-binding material on Superdex 75. The activity was eluted in the volume corresponding to a MW of 54 kDa. SDS–PAGE of this preparation revealed a single band, also corresponding to a 54 kDa protein. MNCF is an acidic protein (pI < 4) as shown by chromatofocussing. Like the crude MNCF, the homogeneous protein induced neutrophil migrationin vitroas well asin vivo. This was not modified by dexamethasone pretreatment.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

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