Ligand binding by murine IgM antibodies: intramolecular heterogeneity exists in certain, but not all, cases.
The ligand binding properties of eight hybridoma-derived murine anti-DNP IgM(kappa) antibodies were analysed by equilibrium dialysis. Four of these proteins exhibited the expected valances of approximately 10 and relatively low affinities (less than or equal to 2.2 x 10(5) M-1). The remaining four proteins exhibited valences of considerably less than 10 (less than or equal to 8) and relatively high affinities (greater than 10(6) M-1). When these proteins were subjected to two cycles of lyophilization, those of the former group were observed to still exhibit approximately 10 sites per molecule with homogeneous affinities similar to those of the respective untreated molecules. However, molecules in the latter group (valences of less than or equal to 8) were observed to exhibit only five to six binding sites subsequent to lyophilization with no changes in affinities. When the reductive subunits from each of the IgM(kappa) proteins were subjected to trypsinization, two different patterns were observed in terms of the yields of Fab mu fragments. Each of the proteins originally exhibiting approximately 10 binding sites yielded greater than 90% of the expected Fab mu fragments. In contrast each of the proteins exhibiting less than or equal to 8 binding sites yielded only approximately 50% of the expected Fab mu fragments. Collectively these results indicate the existence of at least two different forms of murine IgM molecules, those with approximately 10 homogeneous, relatively stable sites and those with only approx. five stable sites. It is suggested that these intramolecular functional differences may be attributable to intramolecular conformational differences.
Pascual
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Department of Microbiology, University of Mississippi Medical Center, Jackson 39216-4505.
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D000818:Animals; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D004591:Electrophoresis, Polyacrylamide Gel; D005362:Ficoll / Q000031:analogs & derivatives / Q000276:immunology; D005612:Freeze Drying; D007075:Immunoglobulin M / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D014357:Trypsin / Q000494:pharmacology
C031007:DNP-Ficoll; D007075:Immunoglobulin M; D005362:Ficoll; D014357:Trypsin