Abstract
A hybridoma line secreting 19S IgM antibodies reactive with the DNP moiety and designated 14PAF was derived from a fusion of murine spleen cells with the myeloma line P3-X63-Ag8. These IgM antibodies exhibited an average of about six homogeneous binding sites per molecule. The failure to demonstrate 10 binding sites per pentameric molecule was attributable to the presence of two different light (L) chains in the secreted molecules. One of the L-chains appeared, based upon limited amino acid sequence studies, to be the L-chain normally found in MOPC-21. the myeloma protein secreted by the P3 myeloma line. The other L-chain, presumably derived from the murine spleen cell used in the fusion. was essential for combining sites for the DNP group. Analysis of the reductive 2μ-2L-chain subunits of the secreted IgM anti-DNP molecules indicated the presence of two types. One type (designated active) absorbed to immunadsorbent columns containing the DNP group, exhibited an average ot two homogeneous binding sites per subunit and did not contain MOPC-21 L-chains. The other type of subunit (designated inactive) did not contain any binding sites for DNP and contained only MOPC-21 L-chains. Recombination studies with mildly reduced and alkylated μ- and L-chains from each type of reductive subunit indicated that the μ-chains were functionally identical. Furthermore these in vitro recombination studies indicated that the noncovalent assembly of the 2μ-2L-chain subunits was influenced by the L-chains in such a way that the recombinant subunits were homogeneous in terms of L-chains. Equilibrium dialysis studies with active homogeneous recombinant subunits indicated the presence of but one binding site per 2μ-2L-chain subunit. These findings implicate the assembly process as a possible important factor in the generation of conformational differences that may account for the intramolecular heterogeneity of ligand binding seen with certain IgM molecules.
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