Immunoglobulin assembly in a mouse myeloma.

Heavy and light chain subunits of immunoglobulin are synthesized on different size classes of polysomes in mouse myelomasl-' and immunized rabbit lymph nodes.4-6 Nascent light chains are released from ribosomes and enter a pool of free light chain which eventually complements with heavy chain, forming the covalently associated immunoglobulin molecule, (HL)2." ' Since the intracellular dimers HL,' H2,8 and L29 have been proposed as synthetic intermediates in different myelomas, the pathway from nascent heavy and light chains to the finished tetramer has remained obscure, particularly with respect to the sequence of interchain disulfide bond formation. Part of the apparent contradiction could result from the experimental use of myeloma tumors with multiple stem lines.10-12 By using an exponentially growing, cloned mouse myeloma tissue culture line, the most probable pathway of immunoglobulin biosynthesis and assembly was defined. Materials and Methods.-The tissue culture line of MOPC2113 was established from a clone by Kengo Horibata.14 The line was maintained in Eagle's fortified medium15 plus 10% horse serum at 370C in 5% CO2, 95% air. The cells had a doubling time of 15 hr and only secreted IgG, immunoglobulin. In some experiments, kappa-light chain secreted from MOPC41"1 was used as a marker. Secreted protein was prepared from the supernatant of a cell suspension after 4 hr in the presence of C-l-eucine as previously described.'0 For amino acid labeling, exponentially growing cells were washed twice and resuspended in Eagle's medium minus leucine at 1 X 107 cells per ml. After 15 min at 370C, the cells were labeled with H'-leucine (45 mc/Mmole, 20 Mc/ml final concentration). For the preparation of polysomes, labeled cells were centrifuged and resuspended in pH 6.7 buffer' (0.025 M phosphate, 0.075 M NaCl, 0.01 M MgCl2) containing 0.25 M sucrose. The cells were lysed with 1% Nonidet P.40,' the nuclei removed by centrifugation at 500 X g for 5 min, and the supernatant fraction layered directly onto a 0.3-1 M sucrose gradient in the same buffer. The gradient was centrifuged at 5C for 150 min (23,000 rpm in the 25.1 head of a Beckman model L-2 ultracentrifuge). OD260 was monitored through a Gilford continuous-flow spectrophotometer. Serological precipitation was done by the indirect procedure. Fractions were diluted fivefold with saline, and 30 ul of rabbit anti-MOPC21 immunoglobulin were added and incubated for 15 min at 370C. An excess of goat anti-rabbit immunoglobulin was then added, and incubation continued at 370C for another hour. After refrigeration overnight, the precipitates were washed three times in cold saline. For nonspecific precipitation, a control of normal rabbit serum was used. All data are expressed as the difference between the two; the backgrounds are given in the figures. The rabbit antiserum was prepared against purified MOPC21 serum immunoglobulin, and reacted with kappa-type light chain, MOPC21 immunoglobulin, and the Fc fragment of normal mouse IgG. Precipitates were dissolved in 0.01 N NaOH, reprecipitated from 10% trichloroacetic acid, collected on Millipore filters, and counted in a scintillation counter. Electrophoresis at pH 7.1 in 7.5% acrylamide, 0.5 M urea, and 0.1% sodium lauryl sulfate (SDS) was performed according to Maizel,'6 and fractions counted according to Choules and Zimm.'7 Serological precipitates were solubilized for electrophoresis by the procedure of Maizel;'6 0.2 M 2-mercaptoethanol was used as a reducing agent when indicated. Electrophoresis was carried out with n internal control of CIabeled s-