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Surface antigens of three stages of the nematode worm Trichinella spiralis has been labelled with iodine and examined by sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. A variety of interactions were defined: the infective larva surface antigens formed a spectrum of aggregates from 50kDa to greater than 1000kDa from subunits of 47kDa and 90kDa; in the adult worms of 60kDa complex arose fron interaction between two dissimilar molecules of 40kDa and 20kDa; the new-born larvae components formed homologous dimers from a 58kDa molecule. Aggregating molecules were adherent to lentil lectin-Sepharose and are therefore glycoproteins. The interactions observed were completely abolished by boiling in SDS/mercaptoethanol, but only partially destroyed by boiling in SDS/iodoacetamide. Based upon this, the associations can be characterized as non-covalent, but disulphide-bond-dependent. It is suggested, but not proved, that the aggregates arise from strong non-covalent hydrophobic interaction sites which are stabilized by intrachain disulphide bonds in the molecules concerned.

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.

Immunoglobulin M (IgM) biosynthesis was studied with mouse plasma-cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with [3H]leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The tumour secretes IgM and light chains. ‘Pulse–chase’ experiments indicated average secretion times of 1.5h for light chain and 2.5h for IgM. The order of disulphide-bond assembly within the cell was shown to be heavy chain+light chain → heavy chain–light chain intermediate → IgMs. The 7S subunit (IgMs) was polymerized into IgM just before or at the time of secretion. Measurements of heavy-chain/light-chain radioactivity ratios in intracellular HL and IgMs and secreted IgM demonstrated the existence of a light-chain pool participating in IgM biosynthesis. The size of the light-chain pool, together with analysis of clones isolated in vivo, suggested that the tumour contains cells in which light-chain synthesis is in excess of heavy-chain production.

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 [3H]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 (5–100μg/ml) and total reconstitution at higher protein concentrations (300μg/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.

Incorporations of radioactive mannose, galactose and fucose into MOPC 104E mouse plasma-cell tumour suspensions suggest a stepwise addition of carbohydrate residues to immunoglobulin M (IgM) during the process of secretion. Mannose and glucosamine residues are added at an early stage, whereas galactose and fucose are added just before, or at the time that, IgM leaves the cell. Free light chains secreted in excess by the same tumour cells incubated with mannose, galactose or fucose contained barely detectable amounts of radioactivity.

Immunoglobulin M biosynthesis was studied with mouse plasma cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with tritiated leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by polyacrylamide-gel electrophoresis, sucrose-density-gradient centrifugation and precipitation with rabbit antiserum specific for the macroglobulin. The tumour was found to secrete immunoglobulin M and light chains in a 1:2 weight ratio, with lag periods of 20–30min. Within the cells there was a 7s component precipitable with specific antiserum to the macroglobulin that was shown to consist of heavy and light chains. This 7s subunit of the macroglobulin appeared to accumulate in the intracellular environment, so that even after long periods of incubation (3hr.) no more than trace amounts of fully assembled 19s molecules could be detected in cell lysates. Polymerization of the subunits into the pentamer therefore appears to take place shortly before, or simultaneously with, secretion of the molecules.

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37°C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.

Cell suspensions of mouse plasma-cell tumour MOPC 315 secreting predominantly IgA (immunoglobulin A) monomer and dimer were incubated with radioactive leucine, mannose, galactose and fucose for various periods of time. The amounts of secreted and intracellular immunoglobulins were measured by co-precipitation with specific antibody, and the molecular species present were assessed by electrophoresis in polyacrylamide gels. Analysis of the secreted myeloma protein demonstrated that monomer and dimer IgA molecules are identical with respect to carbohydrate composition and rate of secretion. Within the cell, the myeloma protein is almost entirely accounted for by monomer units which either leave the cell as such or are polymerized with the addition of J chain close to the time of secretion. The results support the concept of a stepwise addition of carbohydrate residues to IgA immunoglobulin during the process of secretion. Similar patterns of carbohydrate assembly were found for the monomer or dimer molecules. Mannose residues are added at an early stage, whereas fucose is added close to the time of secretion. Galactose is also added early, but some may also be incorporated at a later stage. Control of IgA polymerization is considered unlikely to reflect regulation at the level of carbohydrate addition, and it is suggested that the critical controlling factor is the J chain.