Determination of dissociation constants and ligand specificity of detergent solubilized surface membrane immunoglobulin A from MOPC-315.
Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.
D000818:Animals; D000918:Antibody Specificity; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D003902:Detergents; D007070:Immunoglobulin A / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D010954:Plasmacytoma / Q000276:immunology; D011947:Receptors, Antigen, B-Cell / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D003902:Detergents; D007070:Immunoglobulin A; D008024:Ligands; D011947:Receptors, Antigen, B-Cell