Production and characterization of monoclonal antibodies against the two subunits proteins B1 and B2 of Escherichia coli ribonucleotide reductase.
Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, named protein B1 (170 000) and protein B2 (87 000). We purified and characterized five monoclonal antibodies against B1 and three against B2 from hybridomas obtained by fusion of spleen cells from immunized mice and the myeloma cell line P3-X63Ag8. All are of the IgG1 class with a high affinity for the antigen with dissociation constants in the nanomolar range. Four of the anti-B1 monoclonals and all three anti-B2 monoclonals neutralize reductase activity while one anti-B1 monoclonal binds tightly to B1 without affecting its activity. Fab fragments prepared from three anti-B1 monoclonals had similar dissociation constants. The anti-B1 monoclonals interacted with separate epitopes while two of the anti-B2 monoclonals appeared to react with the same epitope. In the case of B1, various allosteric states of the protein induced by binding of effectors had no apparent effect on the interaction with monoclonals, nor did their binding prevent subsequent binding of effectors. With B2, binding of monoclonals did not affect the typical electron paramagnetic resonance spectrum of the protein and thus did not involve either the tyrosyl free radical or the iron center of B2. All neutralizing antibodies interfered with the interaction between the two subunits, explaining their effect on enzyme activity, since active ribonucleotide reductase consists of a B1-B2 complex.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004926:Escherichia coli / Q000201:enzymology*; D005260:Female; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D011863:Radioimmunoassay; D012264:Ribonucleotide Reductases / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D012264:Ribonucleotide Reductases