scholarly journals Immunochemical characterization of human plasma fibronectin

1980 ◽  
Vol 191 (3) ◽  
pp. 719-727 ◽  
Author(s):  
M Vuento ◽  
E Salonen ◽  
K Salminen ◽  
M Pasanen ◽  
U K Stenman
Keyword(s):  
Human Plasma ◽  
Antigenic Structure ◽  
Antigenic Determinants ◽  
Proteolytic Degradation ◽  
Plasma Fibronectin ◽  
Native Protein ◽  
Covalent Interaction ◽  
Antigenic Activity ◽  
Haemagglutinating Activity

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331–337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000–200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.

Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin

1984 ◽  
Vol 790 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Yu Yamaguchi ◽  
Mamoru Isemura ◽  
Masashi Kosakai ◽  
Akira Sato ◽  
Masakuni Suzuki ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasma Fibronectin ◽  
Adult Plasma

scholarly journals Primary structure of human plasma fibronectin. Characterization of a 38 kDa domain containing the C-terminal heparin-binding site (Hep III site) and a region of molecular heterogeneity

1987 ◽  
Vol 241 (3) ◽  
pp. 923-928 ◽  
Author(s):  
A Garcia-Pardo ◽  
A Rostagno ◽  
B Frangione
Keyword(s):  
Human Plasma ◽  
Primary Structure ◽  
Terminal Segment ◽  
Molecular Heterogeneity ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Heparin Binding Site ◽  
Heparin Binding Domain ◽  
Polypeptide Chains

The primary structure of a 38 kDa heparin-binding domain from human plasma fibronectin has been determined. This domain contains 380 residues arranged in three type-III homology regions of approx. 90 residues each, and a 67-amino-acid C-terminal segment. This segment has been shown to be encoded by certain mRNA species only, due to alternative splicing [Kornblihtt, Vibe-Pedersen & Baralle (1984) Nucleic Acids Research 12, 5853-5868], and therefore represents a region of heterogeneity in fibronectin. Our data indicate that at least one of the constituent polypeptide chains contains this region.

Production and Characterization of Monoclonal Antibodies Directed against the Laminin Receptor Precursor

1997 ◽  
Vol 12 (1) ◽  
pp. 1-5 ◽  
Author(s):  
S. Butò ◽  
C. Ghirelli ◽  
P. Aiello ◽  
E. Tagliabue ◽  
E. Ardini ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Monoclonal Antibodies ◽  
The Other ◽  
Laminin Receptor ◽  
Antigenic Determinants ◽  
Native Protein ◽  
Partially Unfolded ◽  
Cross Competition ◽  
Membrane Level

The 67-kDa laminin receptor (67LR) is an important tumor marker whose molecular structure has not yet been fully elucidated. To shed new light on this molecule, we raised a series of eight new monoclonal antibodies, designated MPLR1 to 8, directed against the 37-kDa recombinant laminin receptor precursor (37LRP). Cross-competition experiments demonstrated that the epitopes recognized by MPLR2, 4 and 5 partially overlap, since MPLR4 and 5 compete with labelled MPLR2 for the binding to recombinant 37LRP. These three antibodies belong to the IgG1 class, whereas the other ones are all IgM. Presumably due to the fact that they are directed against partially unfolded antigenic determinants expressed on the recombinant protein, MPLRs did not recognize the native protein. Indeed, they showed no reactivity at the membrane level in cytofluorimetric analysis and they did not work in immunoprecipitation experiments. In contrast, these reagents are valuable tools in immunoblotting, since they clearly identify a 67-kDa protein (the mature laminin receptor) in addition to the 37-kDa precursor form. MPLRs are thus a new powerful tool which could help in the characterization of the still enigmatic 67LR molecule.

Immunochemical Studies of Diphtherial Toxin and Related Nontoxic Mutant Proteins

1980 ◽  
Vol 30 (3) ◽  
pp. 835-846
Author(s):  
Stanley J. Cryz ◽  
Susan L. Welkos ◽  
Randall K. Holmes
Keyword(s):  
Enzymatic Activity ◽  
Binding Sites ◽  
High Titer ◽  
Binding Activity ◽  
Antigenic Structure ◽  
Antigenic Determinants ◽  
Mutant Proteins ◽  
Neutralizing Activity ◽  
Formaldehyde Treatment

Competitive binding radioimmunoassays were used to analyze the immunochemistry of diphtherial toxin. Rabbit antisera obtained by immunization with formolized toxoid or fragment A were used to characterize purified toxin, toxoid, fragment A, and related nontoxic mutant proteins. Antitoxoid serum had a high titer of neutralizing activity. Most of the antibodies in antitoxoid bound to toxin but not to fragment A. The anti-fragment A antibodies that were present in antitoxoid recognized determinants of fragment A that were exposed on unnicked toxin. Formaldehyde treatment partially destroyed antibody-binding sites associated with the A and B domains of toxin. Anti-fragment A serum had a low titer of neutralizing activity. The specificities of the anti-fragment A antibodies in antitoxoid and anti-fragment A sera were different. Approximately half of the anti-fragment A antibodies in anti-fragment A serum recognized determinants of fragment A that were masked in toxin. Per unit of fragment A-binding activity, anti-fragment A serum was significantly more potent than antitoxoid serum as an inhibitor of the enzymatic activity of fragment A. By analyzing the antigenic structure of several nontoxic mutant proteins (cross-reacting materials) that cross-react with toxin, we distinguished three different subgroups of antigenic determinants associated with the B domain of toxin. Furthermore, the exposed antigenic determinants of the A domain of toxin were separated into two subgroups, both of which were distinct from the masked determinants of the A domain. The radioimmunoassays described here provide rapid, sensitive, quantitative, and versatile methods for immunochemical characterization of toxin or related cross-reacting proteins encoded by corynebacteriophages.

scholarly journals Isolation and characterization of rat plasma fibronectin

1981 ◽  
Vol 197 (3) ◽  
pp. 529-534 ◽  
Author(s):  
R E Weiss ◽  
A H Reddi
Keyword(s):  
Proteinase Inhibitors ◽  
Rat Plasma ◽  
Proteolytic Degradation ◽  
Affinity Column ◽  
Plasma Fibronectin ◽  
Collagen Binding ◽  
Isolation And Characterization ◽  
Monospecific Antibodies ◽  
Shed Light

Rat plasma fibronectin has been isolated and characterized and monospecific antibodies were prepared to it. Two components of fresh rat plasma (in the presence of proteinase inhibitors) bound to a gelatin-Sepharose affinity column. One protein was eluted with 4.0 M-urea and was identified as fibronectin. Another protein was eluted from the gelatin-Sepharose column with 8.0 M-urea and was identified as a 70 000-Mr collagen-binding molecule. This 70 000-Mr fragment was found to be a normal constituent of blood plasma, and its presence did not represent a proteolytic degradation product formed during isolation. The antibodies prepared against rat fibronectin only weakly cross-reacted with plasma fibronectins of chicken, horse and human. These studies shed light on the metabolic interrelationships between fibronectin and other collagen-binding molecules.

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