THE STRANGE CROSS-REACTION OF MENADIONE (VITAMIN K3) AND 2,4-DINITROPHENYL LIGANDS WITH A MYELOMA PROTEIN AND SOME CONVENTIONAL ANTIBODIES

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of [3H]-ε-DNP-L-lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K3). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 105 L/M (at 4°C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal)

A Current Look at the Biological Basis of Antibody Diversity and Specificity

Using automated protein sequence analysis of BALB/c myeloma proteins, the genetic basis for antibody specificity and diversity is investigated. Studies on the N-terminal regions of the heavy chains from these immunoglobulins reveal that a large amount of diversity must exist in the VH regions. Examination of the heavy and light chain sequences from myeloma proteins with hapten-binding activities indicates that the heavy chain variable region sequence correlates closely with all the specificities of intact molecules. The light chain appears to be less restricted in some specificities, however. The relevance of these data to the proposed mechanisms of antibody diversity is discussed

Antibody Diversity

Vertebrate organisms possess a large and diverse repertoire of antibody variable regions. A number of different genetic mechanisms have been proposed to account for immunoglobulin variable (V) region diversity, including multiple germline genes, somatic mutation, somatic recombination, and multiple small gene segments which are joined to form a complete variable region gene segment. Analyses of variable region amino acid sequences demonstrate the relative contribution of each of these mechanisms to antibody diversity. Twenty-four VK21 chains have been examined. They suggest that the kappa chain variable region is encoded in two separate gene segments: VK and JK which are rearranged and joined during B cell differentiation. Diversification of the N terminus of the JK segment occurs as a consequence of VK-JK joining and has been explained by a site-specific recombination model. The amino acid sequence data are consistent with the existence of a minimum of six VK and five JK germline gene segments. Possible cases of somatic mutation are also observed. These conclusions are supported by nucleic acid sequence analyses performed by others. Complete variable region amino acid sequences have been determined for twenty-one heavy chains from dextran binding antibodies. These sequences suggest that the heavy chain variable region is encoded by three gene segments: VH, D, and JH. Nucleic acid sequence analyses are consistent with this conclusion. The existence of a minimum of two VH and four JH germline gene segments is suggested by these sequences. Possible examples of somatic mutation of VH and JH gene segments have also been found. Diversification of the N-terminal residue of the JH segment may occur as a consequence of D-JH joining by a mechanism analogous to that observed in kappa chains. Although comprised of only two residues, the D segment is the most diverse portion of dextran binding heavy chains. Combinatorial joining of VK and JK gene segments and VH, D, and JH gene segments contributes significantly to antibody diversity. Precise molecular locations of idiotypic determinants can be established in the dextran heavy chains. A cross-reactive idiotypic determinant (IdX) is located in the second hypervariable region of the VH segment. Individual idiotypic determinants (IdIs) correspond to particular D segments.</p