Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits.
We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [(3)H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5-100mug/ml) and total reconstitution at higher protein concentrations (300mug/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion.
D000477:Alkylating Agents; D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000032:analysis; D004229:Dithiothreitol; D004589:Electrophoresis, Disc; D007074:Immunoglobulin G / Q000378:metabolism*; D007136:Immunoglobulins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D066298:In Vitro Techniques; D007461:Iodoacetates; D007930:Leucine / Q000378:metabolism; D051379:Mice; D009101:Multiple Myeloma / Q000378:metabolism; D009374:Neoplasms, Experimental / Q000378:metabolism; D010084:Oxidation-Reduction; D011108:Polymers; D014316:Tritium
D000477:Alkylating Agents; D004220:Disulfides; D007074:Immunoglobulin G; D007136:Immunoglobulins; D007461:Iodoacetates; D011108:Polymers; D014316:Tritium; D007930:Leucine; D003545:Cysteine; D004229:Dithiothreitol