scholarly journals Immunoprecipitation of the lutropin receptor. Loss of receptor molecules during down-regulation

1984 ◽  
Vol 224 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M K Metsikkö ◽  
H J Rajaniemi
Keyword(s):  
Gamma Globulin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Down Regulation ◽  
Gonadotropin Receptor ◽  
Reducing Conditions ◽  
Lutropin Receptor ◽  
Rat Ovary ◽  
Triton X ◽  
Receptor Antibodies

Specific anti-(lutropin receptor) antibodies were produced by immunizing rabbits with lutropin receptor purified from pseudopregnant rat ovary. The anti-receptor serum at 1:100 dilution together with anti-(rabbit gamma-globulin) serum immunoprecipitated 70% of 3H-labelled, purified lutropin receptor and 42% of 125I-chorio-gonadotropin-receptor complex. The antiserum inhibited hormone binding to rat ovarian particles. Pseudopregnant rat ovarian particles were labelled with periodate/NaB3H4 and solubilized with Triton X-100. The Triton X-100 extract was subjected to immunoprecipitation using the anti-receptor serum. When the immunoprecipitate was dissolved and analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate under reducing conditions followed by fluorography, a receptor polypeptide with an apparent Mr 95000 was detected. A receptor down-regulating dose of choriogonadotropin was injected into pseudopregnant rats and their ovaries were removed and homogenized 4 days later, and analysed for immunoprecipitable receptors as above. No receptor molecules were found. Accordingly, the lutropin receptor molecules actually disappear rather than merely become masked from hormone during homologous down-regulation.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Solubilization and immunoprecipitation of 125I-labelled antigens from Plasmodium knowlesi schizont-infected erythrocytes using non-ionic, anionic and zwitterionic detergents

Parasitology ◽  
1984 ◽  
Vol 88 (1) ◽  
pp. 27-36 ◽  
Author(s):  
R. J. Howard ◽  
J. W. Barnwell
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Plasmodium Knowlesi ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Ionic Detergents ◽  
Class A ◽  
Anionic Detergents ◽  
Triton X

SUMMARYPlasmodium knowlesi malaria-infected erythrocytes were radio-iodinated and several non-ionic, anionic and zwitterionic detergents were compared in their capacity to extract the labelled membrane proteins. The use of these detergents for antigen identification was tested by immunoprecipitation, after addition of Triton X-100 to some detergent extracts, using hyperimmune monkey antiserum and protein A-Sepharose. 125I-labelled antigens were specifically immunoprecipitated with all detergents tested, including the anionic detergents sodium dodecyl sulphate (SDS), deoxycholate and cholate; the zwitterions Zwittergent-312 and -314, CHAPS and Empigen BB, as well as several non-ionic detergents. The SDS-polyacrylamide gel electrophoresis patterns of 125I-labelled antigens varied after extraction with different detergents, there being no consistent pattern for detergents of a particular class. A total of 14 125I-labelled antigens were identified, 11 of them using Triton X-100. Some minor antigens identified with Triton X-100 were immunoprecipitated in greater amount after extraction in other detergents. Most importantly, two antigens Mr 200000 and 180000 were detected only after extraction with deoxycholate or SDS.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Extraction and properties of hemagglutinin from cell wall fragments of Fusobacterium nucleatum

1982 ◽  
Vol 152 (1) ◽  
pp. 298-305
Author(s):  
P Dehazya ◽  
R S Coles
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fusobacterium Nucleatum ◽  
Blue Staining ◽  
Sephadex Column ◽  
Dodecyl Sulfate ◽  
Triton X

To study the hemagglutinin of Fusobacterium nucleatum, methods were sought to solubilize and purify this component. When cells of F. nucleatum were ruptured by passage through a French press, the fragments lost virtually all ability to agglutinate human erythrocytes. Extraction of the fragments with 2% Triton X-100 for 30 min at 22 degrees C restored hemagglutinating activity (HA). Hemagglutination by these fragments could be inhibited by arginine, as can hemagglutination by intact bacteria. Treatment of active cell wall fragments with pronase and 2% Triton X-100-EDTA at 37 degrees C or with pronase and 0.1% Triton X-100-EDTA at pH 10.0 allowed recovery of solubilized HA. The former HA was inhibited by arginine (arg+) whereas the latter was not (arg-). Fractionation of the arg+ extract by preparative isoelectric focusing showed that HA was recovered from the gel sections having a pH between 4.5 and 5.5. Hemagglutination by this preparation was still arg+. Chromatography of this hemagglutinin on DEAE-Sephadex increased the specific activity to high levels with a loss of inhibition by arginine. A fraction from the DEAE-Sephadex column containing 10,700 HA units per mg of protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Solubilization at 22 degrees C before electrophoresis revealed three Coomassie blue-staining bands which migrated with apparent molecular weights of about 21,000, 38,000 and 60,000. When the same DEAE fraction was boiled in sodium dodecyl sulfate, electrophoresis revealed only one band with an apparent molecular weight of 21,000.

scholarly journals Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 9-18 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Mitochondria ◽  
Exchange Chromatography ◽  
Glycerophosphate Dehydrogenase ◽  
Pig Brain ◽  
Purification And Properties ◽  
High Concentrations ◽  
Triton X

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

Cross-linking of neuropeptide Y to its receptor on rat brain membranes

1989 ◽  
Vol 256 (3) ◽  
pp. G637-G643 ◽  
Author(s):  
P. J. Mannon ◽  
I. L. Taylor ◽  
L. M. Kaiser ◽  
T. D. Nguyen
Keyword(s):  
Neuropeptide Y ◽  
Rat Brain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Brain Membranes ◽  
Npy Receptor ◽  
Rat Brain Membranes ◽  
Immature Form ◽  
Triton X

The receptor for neuropeptide Y (NPY) was identified on rat brain membranes after covalent labeling with 125I-NPY using the homobifunctional cross-linkers disuccinimido suberate and disuccinimido dithiobis(propionate) and the heterobifunctional photoactive cross-linker succinimido 4-azidobenzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed the presence of two bands at Mr 62,000 and 39,000. Both species showed the same high affinity for 125I-NPY. Exposure to reducing agents did not change the migration of these bands. When the NPY receptor complex was solubilized from the membranes with 1% Triton X-100 and analyzed by gel filtration chromatography, it eluted from a Fractogel TSK 55F column as a peak at approximately 65 kDa. This peak was asymmetric with a shoulder of radioactivity that probably reflects the smaller receptor species. These data indicate that the NPY receptor on rat brain membranes is a monomeric 58-kDa unit (62 kDa minus the mass of the cross-linked NPY) without covalently or noncovalently linked subunits. The smaller 39-kDa species may be an immature form of the 62-kDa species, a second distinct receptor, or a degradation product of the 62-kDa band.

Atrial natriuretic peptide receptors in renal papilla of DOCA-salt hypertensive rats

1990 ◽  
Vol 259 (1) ◽  
pp. F130-F137
Author(s):  
E. Nuglozeh ◽  
G. Gauquelin ◽  
R. Garcia ◽  
J. Tremblay ◽  
E. L. Schiffrin
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasma Renin ◽  
Renal Papilla ◽  
Hypertensive Rats ◽  
Reducing Conditions ◽  
Anp Receptors ◽  
Atrial Natriuretic

The receptor for atrial natriuretic peptide (ANP) in the rat renal papilla was characterized pharmacologically. After solubilization and irreversible binding with disuccinimidylsuberate, it was shown on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to be made of a single peptide of 125 kDa. The regulation of the renal papillary ANP receptor was studied in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. DOCA-salt rats had suppressed plasma renin activity and increased plasma ANP concentrations (408 +/- 35 vs. 133 +/- 12 pg/ml in uninephrectomized controls, P less than 0.01). The renal papilla was hypertrophied in DOCA-salt hypertensive rats (93 +/- 1 vs. 52 +/- 1 mg, P less than 0.01). The density of ANP sites in the papilla was significantly higher in DOCA-salt rats (141 +/- 31 fmol/papilla) than in controls (34 +/- 8 fmol/papilla, P less than 0.01). Affinity of sites in DOCA-salt rats and controls was similar. The production of guanosine 3',5'-cyclic monophosphate (cGMP) in renal papilla in response to ANP was significantly higher in DOCA-salt rats. In contrast to the renal papillary ANP receptor, acid-washed vascular and glomerular ANP sites were significantly decreased in density in DOCA-salt hypertensive rats. In blood vessels and glomeruli, both the high- and low-molecular mass receptor (as detected on SDS-PAGE under reducing conditions) was proportionately decreased in density in DOCA-salt hypertensive rats. The present results suggest that an increased number of ANP receptors and exaggerated cGMP response to ANP in the renal papilla may underlie the increased natriuretic responsiveness of the kidney to ANP in DOCA-salt hypertensive rats.

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity

1990 ◽  
Vol 36 (1) ◽  
pp. 33-41 ◽  
Author(s):  
James E. Piechura ◽  
Viswanth P. Kurup ◽  
Laureen J. Daft
Keyword(s):  
Aspergillus Fumigatus ◽  
Protease Activity ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Allergic Bronchopulmonary Aspergillosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Weights ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73 000 and 43 000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin–avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that the two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases. Key words: Aspergillus, membrane, allergens, proteases, aspergillosis.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Glutathione-s-transferase is an important antigen in the eel nematode Anguillicola crassus

1997 ◽  
Vol 71 (4) ◽  
pp. 319-324 ◽  
Author(s):  
M.E. Nielsen ◽  
K. Buchmann
Keyword(s):  
Specific Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anguilla Anguilla ◽  
European Eel ◽  
Glutathione S Transferase ◽  
Elisa System ◽  
Humoral Immune ◽  
Anguillicola Crassus ◽  
Reducing Conditions

AbstractDifferent organs and secretions/excretions of the swimbladder parasite, Anguillicola crassus (Nematoda), were tested for the presence of antigens to the humoral immune response previously detected in the European eel, Anguilla anguilla. Proteins from different fractions of Anguillicola crassus were separated using SDS–PAGE (sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis) under reducing conditions and electroblotted onto nitrocellulose membranes. Infected eels showed a specific antibody response to a 43 kDa antigen in the cuticle and towards two gonad antigens around 34 and 43 kDa. In protein released from the worms, two secretory/excretory antigens of approximately 28 kDa were found. The secretion/excretion rate of protein from the parasite to the surroundings was determined. Subsequently, an ELISA system was established applying these antigens as the first layer of coating. Furthermore, antigens from Anguillicola crassus were examined for the presence of glutathione-s-transferase (GST) using a specific antibody against GST. The antigens were found to be subunits of GST.

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