Ligand binding and physicochemical characteristics of an IgM mouse plasmacytoma ABPC-22

Michael R. Jarvis, Edward W. Voss

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Immunoglobulin IgM22, secreted by mouse plasmacytoma ABPC-22, was characterized on the basis of ligand binding and physicochemical properties. Upon purification, only two polypeptides (μ aqnd κ chains) were detected by 6 M urea isoelectric focusing in polyacrylamide and SDS-polyacrylamide electrophoresis (SDS-PAGE). The μ and κ chains possessed pI values of 6.27 and 6.13 respectively, in 6 M urea. The molecular masses of μ and κ chains were established by SDS-PAGE and molecular sieve chromatography to be 70 and 30 kd, respectively. Thus, a molecular weight of 1 × 106 was estimated for non-reduced IgM22, and was confirmed by molecular sieve chromatography and sedimentation equilibrium ultracentrifugation. J chain was detected in freshly purified samples. As shown by competitive hapten inhibition (CHI) of the precipitation assay, using 125I-rhodamine B5BSA as the reference ligand, IgM22 bound a wide range of structurally related and unrelated probes. Competing for the same site were polyaromatic anions (such as pyrene-1-sulfonate, rhodamine B and fluorescein) and weakly inhibitory organic acids (such as acetate and propionate). In addition. Fab fragments inhibited precipitation of 125I-rhodamine B5BSA by IgM22. Equilibrium dialysis studies revealed a Ka f 1.5 × 103 M-1 for the interaction of 14C-fluorescein with IgM22. A Ka of 3.2 × M-1 for rhodamine B was determined using a molecular sieve column equilibrated in varying concentrations of rhodamine B. Based on relative Ka values, CHI results and fluorescence binding data with anilinonaphthalene sulfonate (ANS), the nature of the antibody active site was described in detail.

Original languageEnglish
Pages (from-to)261-275
Number of pages15
JournalMolecular Immunology
Issue number4
StatePublished - Apr 1981


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