Assessment by sedimentation equilibrium analysis of a heterologous macromolecular interaction in the presence of self-association: interaction of S5 with S8

Biochemistry ◽  
1981 ◽  
Vol 20 (17) ◽  
pp. 4861-4866 ◽  
Author(s):  
Stephen H. Tindall ◽  
Kirk C. Aune
Keyword(s):  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Self Association

Sedimentation Equilibrium Analysis of ClpB Self-Association in Diluted and Crowded Solutions

2015 ◽  
pp. 135-160 ◽  
Author(s):  
Carlos Alfonso ◽  
Urko del Castillo ◽  
Ianire Martín ◽  
Arturo Muga ◽  
Germán Rivas
Keyword(s):  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Self Association

scholarly journals Studies on the antigenic determinants in the self-association of IgG rheumatoid factor

1981 ◽  
Vol 154 (1) ◽  
pp. 112-125 ◽  
Author(s):  
FA Nardella ◽  
DC Teller ◽  
M Mannik
Keyword(s):  
Sedimentation Equilibrium ◽  
Antigenic Determinants ◽  
Fab Fragment ◽  
Fab Fragments ◽  
Polymer Formation ◽  
Igg Rheumatoid Factor ◽  
Normal Human ◽  
Self Association ◽  
Equilibrium Ultracentrifugation ◽  
Antigen Antibody

The number, location, and other characteristics of the antigenic determinants for self-association of IgG-rheumatoid factors (IgG-RF) were examined using the IgG-RF isolated from the plasma of one patient as a model system. Affinity chromatography was employed for isolation of the IgG-RF. Sedimentation equilibrium ultracentrifugation was used to study the various interactions. The antigenic valence of IgG-RF Fc, normal human Fc, and rabbit Fc fragments was two for the interaction with Fab fragments from IgG-RF, as might be expected from the molecular symmetry of IgG. The antigenic valence of intact normal IgG, however, was only one, indicating that when one of the available antigenic determinants interacted with the Fab fragment of IgG-RF, the other determinant becomes sterically inaccessible. Reduction and alkylation, known to increase the flexibility of the hinge region, did not alter the antigenic valence of IgG for Fab fragments of IgG-RF. The antigenic valence of IgG-RF in self-association could not be experimentally determined but must be two to permit the observed concentration-dependent further polymer formation of IgG-RF dimers. Unique antigenic determinants on the Fc fragments of IgG-RF were sought and not found, thus reaffirming the formation of two antigen-antibody bonds as the basis for dimerization of IgG-RF molecules. The pFc' and Fc' fragments, representing Cγ3 domains of IgG, failed to show significant interaction with Fab fragments of IgG-RF, indicating that the antigenic determinants were not expressed by the Cγ3 regions but are located either on Cγ2 region or require intact Cγ2 and Cγ3 regions for expression. These conclusions were corroborated by the antigenic valence of one for the Fc(i) fragment, a new papain-generated intermediate fragment of Fc, composed of two intact Cγ3 domains and one intact Cγ2 domain. Normal IgG, because of its valence of one for interaction with IgG-RF, would effectively terminate further polymerization of IgG-RF dimers. This may well in part explain the finding of smaller IgG-RF complexes in the serum than in synovial fluid of patients with rheumatoid arthritis.

scholarly journals The structure of L-lactate oxidase from Mycobacterium smegmatis

1977 ◽  
Vol 165 (2) ◽  
pp. 375-383 ◽  
Author(s):  
P A Sullivan ◽  
C Y Soon ◽  
W J Schreurs ◽  
J F Cutfield ◽  
M G Shepherd
Keyword(s):  
Electron Microscopy ◽  
Mycobacterium Smegmatis ◽  
Sedimentation Velocity ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Velocity Analysis ◽  
Approach To Equilibrium ◽  
Lactate Oxidase ◽  
Dimethyl Suberimidate

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.

scholarly journals Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

1985 ◽  
Vol 229 (2) ◽  
pp. 419-428 ◽  
Author(s):  
T K S Mukkur ◽  
D L Watson ◽  
K S Saini ◽  
A K Lascelles
Keyword(s):  
Small Intestine ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Goblet Cells ◽  
Immunofluorescence Assay ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Total Carbohydrate ◽  
Protamine Sulphate

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

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