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.