scholarly journals Purification and some properties of rabbit antiovalbumin

1973 ◽  
Vol 135 (4) ◽  
pp. 705-711 ◽  
Author(s):  
Aftab A. Ansari ◽  
A. Salahuddin
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acid Media ◽  
Stokes Radius ◽  
Antigen Antibody ◽  
Two Peaks ◽  
Complete Dissociation ◽  
Phosphate Groups

Unlike previous reports that the ovalbumin–anti-ovalbumin complex did not dissociate completely in acid media, our results showed complete dissociation of the complex both in 1.2m-acetic acid, pH2.3, and in KCl–HCl, pH2.2, I 0.06. Thus Sephadex chromatography of the solution obtained by dissolving the antigen–antibody precipitate in these media repeatedly gave two peaks corresponding to anti-ovalbumin and ovalbumin. Further, gel-diffusion and immunoelectrophoresis experiments showed that the phosphate groups of ovalbumin are not involved in the antigenic sites. The antibody thus purified was more easily precipitated than previous preparations. The molecular weight and Stokes radius of the antibody were calculated from its gel-filtration behaviour and were found to be 148000 and 4.8nm respectively. The molecular weight determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis was essentially similar (about 0.7% lower).

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 Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

Purification and partial characterization of a plasma inhibitor of tonin

1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Angiotensin I ◽  
Stokes Radius

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin

scholarly journals New Exfoliative Toxin Produced by a Plasmid-Carrying Strain of Staphylococcus hyicus

1999 ◽  
Vol 67 (8) ◽  
pp. 4014-4018 ◽  
Author(s):  
Hisaaki Sato ◽  
Takao Watanabe ◽  
Yasuko Murata ◽  
Ayumi Ohtake ◽  
Mayumi Nakamura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
The Other ◽  
Exfoliative Toxin ◽  
Sds Page ◽  
Serotype B

ABSTRACT A new serotype of Staphylococcus hyicus exfoliative toxin (SHET), serotype B, was isolated from the culture filtrate of a plasmid-carrying strain of S. hyicus. The new SHET was purified by precipitation with 70% saturated ammonium sulfate, gel filtration on a Sephadex G-75 column, column chromatography on DEAE–Cellulofine A-500, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The new SHET caused exfoliation of the epidermis as determined by the so-called Nikolsky sign when inoculated into 1-day-old chickens. The new SHET was serologically different fromStaphylococcus aureus exfoliative toxins (ETs) (ETA, ETB, and ETC) and from the SHET from the plasmidless strain but showed the same molecular weight as the other serotypes of toxins on SDS-PAGE. It was thermolabile and lost its toxicity after being heated at 60°C for 30 min. We propose that the new SHET be designated SHETB and that the SHET produced by the plasmidless strain be designated SHETA.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content

1980 ◽  
Vol 189 (1) ◽  
pp. 111-124 ◽  
Author(s):  
N D Light ◽  
A J Bailey
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Type I Collagen ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Type I ◽  
Molecular Weight Range ◽  
Carbohydrate Analysis ◽  
Weight Range

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.

scholarly journals Bovine lens aldehyde dehydrogenase. Purification and preliminary characterization

1983 ◽  
Vol 215 (2) ◽  
pp. 351-359 ◽  
Author(s):  
H H Ting ◽  
M J C Crabbe
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Bovine Lens ◽  
Systemic Corticosteroids ◽  
Apparent Homogeneity ◽  
Stokes Radius ◽  
Equilibrium Ultracentrifugation

Cytoplasmic aldehyde dehydrogenase from bovine lens was purified to apparent homogeneity by using ion-exchange and affinity chromatography. Sedimentation-equilibrium ultracentrifugation, gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis show that the enzyme is a dimer of Mr 114000, with subunits of Mr 57000. The enzyme does not dissociate into monomers in the presence of Ca2+ or Mg2+. The enzyme has a pI of 5.0, an activation energy of 35.1kJ/mmol and a pK value of 8.6 with acetaldehyde as substrate. The enzyme is a prolate ellipsoid with a Stokes radius of 4nm. Progesterone, deoxycorticosterone and chlorpropamide inhibited enzyme activity, and this inhibition may play a role in cataract formation in patients maintained on systemic corticosteroids and in tablet-dependent diabetics.

scholarly journals Purification and photoaffinity labelling of a rat cytosolic binding protein specific for 3-methylcholanthrene

1987 ◽  
Vol 242 (2) ◽  
pp. 375-381 ◽  
Author(s):  
P S Arnold ◽  
R C Garner ◽  
B Tierney
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Binding Activity ◽  
Liver Protein ◽  
Photoaffinity Labelling ◽  
High Affinity ◽  
Apparent Homogeneity ◽  
Stokes Radius

Rat hepatic cytosolic proteins which sediment at 4-5 S on sucrose gradients exhibit high-affinity saturable binding for the carcinogen 3-methylcholanthrene. A rat liver protein of Stokes' radius 3 nm, Mr by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 39,000 and with specific 3-methylcholanthrene-binding activity sedimenting at 4.5 S, has been purified 315-fold to apparent homogeneity by using affinity chromatography on a column of 1-hydroxy-3-methylcholanthrene coupled to epoxy-activated Sepharose 6B, in conjunction with two gel-filtration steps. The protein purified by this technique was shown to be associated with the observed specific 3-methylcholanthrene-binding activity by photoaffinity labelling with 1-oxo-3-methylcholanthrene.

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