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 NaDodSO^?₄-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.