Isolation of the monomeric subunit of immunoglobulin M with its interchain disulfide bonds intact

Morris, James E.; Inman, F. P.

doi:10.1021/bi00848a022

Cited by 67 publications

(21 citation statements)

References 15 publications

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“…Assays of the 14C content of the remaining intact monomers showed that 5% had one g-is intrasubunit bond cleaved. These values were consistent with results obtained in similar studies (14). The numbers of alkylated half-cystines contributed by the breakage of slight and i-is intrasubunit bonds were then subtracted from the total [14C]-carboxymethylcysteine content of the A chains to obtain the number of alkylated half-cystines resulting from the cleavage of intersubunit bonds (Table 3).…”

Section: Methodssupporting

confidence: 86%

Mechanism of IgM Polymerization

Chapuis

Koshland

1974

Proc. Natl. Acad. Sci. U.S.A.

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The stoichiometry of J chain in pentamer 1gM has been determined by measuring the radiolabeled thiols in the constituent chains after complete reduction and alkylation of the polymer. One mole of J was found to be disulfide bonded to 1 mol of pentamer. The polymerization of the immunoglobulins presents an intriguing problem because the process determines both the secretion of IgA and IgM polymers from cells and their specialized biological properties after secretion. An understanding of the mechanism of polymerization is, therefore, a necessary step for understanding the biological significance of the polymers. The first clue was obtained when a small polypeptide, the J chain, was found to be disulfide bonded to the polymerized IgA and IgM species, but absent from all monomeric species (1, 2). The linkage role of the J chain has been confirmed in a series of subsequent investigations. For example, biosynthetic studies showed that J and the IgA or IgM monomers are synthesized in the same cell and the polymer is assembled just prior to its secretion (3,4). Recombination experiments demonstrated that J is not merely associated with the polymers, but is necessary for correct assembly (5). Stoichiometric measurements on IgA established that each polymer, independent of its size contains, and therefore requires, only 1 molecule of J chain (6).On the basis of these data, two different mechanisms of polymerization have been postulated (7). In the "bracelet" model J provides an extended backbone to which each monomer subunit is disulfide bonded. In the "clasp" model J is disulfide bonded to only two adjacent monomers and induces direct S-S bonding between the other monomers. To distinguish these mechanisms, a method of stepwise reduction and differential radioalkylation was developed which permitted the cleavage of J chain disulfides to be correlated with the release of polymer subunits. The present paper describes this method and its application to solving the mechanism of IgM polymerization. MATERIALS AND METHODSPurification of IgM. A human Waldenstroms macroglobulin was purified by repeated euglobulin precipitation (8) followed by filtration through a Sepharose 6B column equilibrated with Tris-saline buffer (0.02 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.02% Na azide). The main protein peak was concentrated by ultrafiltration (Diaflo UM-10 membrane). The final preparation contained only pentamers and a few percent larger aggregates as measured by velocity sedimentation. J chain isolated from this IgM preparation by preparative polyacrylamide gel electrophoresis (7) (10), and the gels were stained using the procedure of Weber and Osborn (11). Alkaline urea electrophoresis was carried out on 4.15% polyacrylamide gels according to the method of Reisfeld and Small (12). The gels were frozen, sliced, and prepared for radioactivity measurements as described previously (7). All values given were corrected for background and cross-contamination between the 14C and 3H counting. Sedimentation analysis was performed ...

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Section: Methodssupporting

confidence: 86%

Mechanism of IgM Polymerization

Chapuis

Koshland

1974

Proc. Natl. Acad. Sci. U.S.A.

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“…The types of disulfide bonds differ in their susceptibility to reductive cleavage. Previous findings that intersubunit bonds could be split with lower concentrations of reducing substance than interchain bonds (1,11,12) were confirmed in this study. Furthermore, it was shown that disulfide bonds linking ^ and ] thains were more resistant than the two other types.…”

Section: Discussionsupporting

confidence: 89%

Disulfide Bonds of Human IgM: Differential Sensitivity to Reductive Cleavage

Kownatzki

1973

Scand J Immunol

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Kownatzki, E. Disulfide Bonds of Human IgM: Differential Sensitivity to Reductive Cleavage. Scand. J. Immunol 2, 197.1. Two Waldenstrom macroglobulins were reduced with increasing amounts of dithiothreitol ranging from 0.25 to 2.0 mM. Disruption of intersubunit, interchain and u'J chain disulfide bonds was measured by determining the amount of IgMs, u, chains and J chains released at a j^ivtn DTT concentration. Intersubunit bonds were most labile, while S-S bonds linking ^^ and J chains were most resistant to reductive cleavage. Kownatzki, Instiiut fiir Immunologie und Serologie, Dr. Eckhard

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“…The determinants might therefore indicate a "polymer specificity" arising when the five subunits combine to form a complete ~/M molecule. But disulfide bonds within each subunit are also split under the conditions used for reduction (25,26,27). This may change the molecular conformation within the subunit with loss of antigenic determinants similar to the behavior of "rE.…”

Section: Discussionmentioning

confidence: 99%

Individual Antigenic Specificity of Human Macroglobulins

Harboe

Solheim

Deverill

1969

The Journal of Experimental Medicine

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The localization of individually specific antigenic determinants on the monoclonal γM Mö was studied in gel diffusion experiments with 19S γM Mö and its 7S subunit. Various types of such determinants were delineated. Some were demonstrable both on the intact macroglobulin molecule and on the subunit. The latter was often unable to precipitate with the corresponding antibody, but combined with it and inhibited precipitation. Immunization of rabbits with purified γMs Mö was the most useful way to obtain antisera demonstrating individually specific antigenic determinants on the subunit by direct precipitation. Still other determinants were apparently destroyed by mild reduction of the γM molecule. Reduction and reassociation of mixtures of two monoclonal γM-globulins resulted in formation of hybrid molecules containing subunits of different origin. Individually specific antisera against the two components were useful for demonstrating hybrid formation. Serological activity, i.e., cold agglutinin activity, was present in the hybrid molecules.

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Isolation of the monomeric subunit of immunoglobulin M with its interchain disulfide bonds intact

Cited by 67 publications

References 15 publications

Mechanism of IgM Polymerization

Mechanism of IgM Polymerization

Disulfide Bonds of Human IgM: Differential Sensitivity to Reductive Cleavage

Individual Antigenic Specificity of Human Macroglobulins

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