Reassembly of immunoglobulin M heavy and light chains in vitro.
Reduced and alkylated monoclonal IgM was fractionated into mu and light (L) chains by gel chromatography in 1N acetic acid. Equimolar mixtures of the chains formed a noncovalently bonded structure in 0.01M sodium acetate buffer, pH 4.1, that had the properties of a half subunit. The latter reassociated into a subunit-like structure after transfer into 0.08M sodium phosphate buffer, pH 7.5. The similarity of the reconstituted IgM subunit (IgMs) to that of the native molecule was established by its physicochemical and immunochemical properties. Comparable products were obtained on reassembly of the alkylated mu and L chains from several other monoclonal IgM. The presence of active binding sites for IgG on subunits reconstituted from the chains of proteins with anti-IgG activity further indicated correct assembly of the mu and L chains. High yields of subunit-like products were also obtained by assembly of mu chains from one protein and L chains from another. Evidence was obtained that L chains of appropriate specificity can substitute for the homologous chain in the formation of the active site. Heterogeneous mixtures of high molecular weight products were generated from mu and L chains that were not alkylated. Reduction and alkylation demonstrated that the products represented polymers of reconstituted IgMs. Significant levels of anti-IgG activity were detected in the polymeric IgM generated from the chains of active proteins by precipitation with aggregated IgG.
D002850:Chromatography, Gel; D006801:Humans; D006824:Hybridization, Genetic; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000032:analysis / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000032:analysis / Q000302:isolation & purification; D011108:Polymers; D014461:Ultracentrifugation; D008258:Waldenstrom Macroglobulinemia / Q000276:immunology
D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D011108:Polymers