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.
AuthorsM R Jarvis, E W Voss Jr
JournalMolecular immunology (Mol Immunol) Vol. 20 Issue 1 Pg. 125-36 (Jan 1983) ISSN: 0161-5890 [Print] ENGLAND
PMID6855776 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)
Chemical References
  • Detergents
  • Immunoglobulin A
  • Ligands
  • Receptors, Antigen, B-Cell
  • Animals
  • Antibody Specificity
  • Chemistry, Physical
  • Chromatography, Affinity
  • Detergents
  • Immunoglobulin A (immunology, isolation & purification, metabolism)
  • Ligands
  • Mice
  • Mice, Inbred BALB C
  • Physicochemical Phenomena
  • Plasmacytoma (immunology)
  • Receptors, Antigen, B-Cell (immunology, isolation & purification, metabolism)
  • Solubility

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