Detection of a membrane-like IgM inside of murine spleen cells

Abstract

In an effort to determine at what stage during intracellular transport the precursor to plasma membrane IgM becomes inserted in membranes, the solubility (in the absence of detergent) of radioactive IgM obtained by incubation of cells with³H-leucine was examined. A population of normal spleen cells of mice were used since a considerable proportion of the IgM biosynthesized is membrane IgM and because synthesis of other Ig classes is minimal. Detergent lysates of labeled cells were treated with Bio-Beads SM-2 to remove the detergent, Nonidet P40. Large aggregates were then separated from smaller aggregates and soluble molecules by ultracentrifugation. Between 30 and 40% of the³H-IgM was associated with the large aggregate fraction. Mouse IgG and partially-reduced MOPC 104E mouse IgM did not aggregate under these conditions. Aggregation of pentameric MOPC 104E IgM was sensitive to detergent. Fetal calf serum at 10% concentration reduced aggregation. The percentage of³H-IgM in the large aggregate fraction did not vary with the length of incubation of cells in the presence of radioactive amino acid between 15 min and 2 hr. The ability of intracellular or plasma membrane Ig (labelled with¹²⁵I) to aggregate was not sensitive to prior reduction of the inter-heavy chain bonds. The results are interpreted as supporting the view that insertion into a lipid bilayer is an early event in the biosynthesis of membrane IgM. It was also found that intracellular IgM shares with membrane IgM an anomalously slow mobility on electrophoresis in polyacrylamide gels containing dodecyl sulfate and that the anomalous mobility was no longer associated with molecules that had been partially reduced.