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Differential reduction of interchain disulphide bonds of mouse immunoglobulin G.

ABSTRACT: The relative lability of the interchain disulphide bonds of mouse G(2a)-myeloma protein 5563 was studied as a function of 2-mercaptoethanol concentration. Analysis of partial-reduction mixtures by polyacrylamide-gel electrophoresis and microdensitometry showed that the disulphide bonds between light and heavy chains are much more susceptible to reduction than the bonds between heavy chains. At a low concentration of 2-mercaptoethanol (10mm) the major dissociable products of mouse immunoglobulin G are heavy-chain dimers and free light chains. These findings contrast with the reported behaviour of rabbit immunoglobulin G, for which the lability of inter-heavy-chain bonds was found to exceed that of the bonds linking light and heavy chains (Hong & Nisonoff, 1965); the relative stability of rabbit immunoglobulin G interchain bonds was confirmed in the present study. Examination of human immunoglobulin G and an immunoglobulin G (gamma2) of guinea pig showed that at least in the majority of molecules, as with mouse immunoglobulin G, the disulphide bonds between light and heavy chains are more susceptible to reduction than the inter-heavy-chain bonds.

SUBMITTER: Williamson AR

PROVIDER: S-EPMC1198754 | biostudies-other | 1968 May

REPOSITORIES: biostudies-other

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Interchain disulphide bridges of mouse immunoglobulin M.

Project description:Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the CMU2 region and the other in the C-terminal region. The inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM.

| S-EPMC1172409 | biostudies-other

Antibody organization on lipid films. Influence of pH and interchain disulphide reduction.

Project description:Two distinct lattice structures are observed for two-dimensional (2-D) antibody organization on phospholipid films. A low-order, small-unit-cell, square lattice is obtained at pH 7 and below for mouse IgE, mouse IgG2a and IgG2b and rabbit IgG. At pH 7.5 and above, the observed lattice structure switches to a large-unit-cell, hexagonal type for rabbit IgG and mouse IgE. Interchain disulphide reduction by exposure to 2 mM-dithiothreitol results in the formation of the compact 2-D lattice for all cases and for all pH conditions.

| S-EPMC1149654 | biostudies-other

The disulphide bonds of the heavy chain of rabbit immunoglobulin G.

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| S-EPMC1185354 | biostudies-other

Interchain disulphide-bond formation in the assembly of immunoglobulin G. Heavy-chain dimer as an intermediate.

Project description:1. The role of disulphide-bond formation in the assembly of G(2a) myeloma protein 5563 was studied by pulse-labelling ascitic plasma cells of tumour-line 5563 for 2-8min. with radioactive amino acids, and analysing the intracellular proteins. Myeloma-protein determinants were first purified by ion-exchange chromatography under conditions that do not dissociate non-covalently linked sub-units of immunoglobulin G. The pulse-labelled material was then analysed by electrophoresis on polyacrylamide gels in sodium dodecyl sulphate-phosphate-urea buffer, which dissociates non-covalently linked sub-units; after gel electrophoresis, radioactive protein bands were located by radioautography, and characterized immunologically after elution. 2. Two heavy-chain intermediates were detected: (i) heavy-chain dimer; (ii) the dimer with one light chain attached. Free light chains had previously been shown to be intermediates in assembly. No evidence for the presence of half-molecules (one light chain attached to one heavy chain) was obtained. The formation of the disulphide bond between the heavy chains thus appears to precede the light-chain-heavy-chain linkage in immunoglobulin G assembly.

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Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.

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The effect of chemical modifications induced in insulin on the reactivity of the interchain disulphide bonds towards sodium sulphite.

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| S-EPMC1198800 | biostudies-other

The disulphide bridges of a mouse immunoglobulin G1 protein.

Project description:[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species.

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Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement.

Project description:Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.

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Project description:The reduction of mixed disulphides of some proteins and GSH [Protein(-SSG)n] is accomplished with GSH as a reductant and a thioltransferase from rat liver as a catalyst, thus: See article. The spontaneous reaction is negligible in comparison with the enzymic reaction in vivo, and any direct reduction with glutathione reductase is not detectable with the substrates used. The reduction is only indirectly dependent on NADPH, which is required for the regeneration of GSH from GSSG. Other protein disulphides apparently are reduced via analogous GSH-dependent reactions

| S-EPMC1165689 | biostudies-other

On the disulphide bonds of rhodopsins.

Project description:Carboxymethylation using 14C- or 3H-labelled iodoacetic acid has been used to identify the cysteine residues in bovine rhodopsin involved in the formation of the two intramolecular disulphide bridges. Iodo[2-14C]acetic acid was used to modify 5.8-5.9 residues of cysteine under non-reducing conditions. After dialysis and reduction of disulphide bridges by 2-mercaptoethanol, iodo[2-3H]acetic acid was employed to covalently modify 3.3-3.6 residues of cysteine. Peptide purification and sequencing has unambiguously shown that cysteine residues 322 and 323 are only carboxymethylated after reduction of disulphide bridges. Indirect evidence presented, now coupled with the earlier finding [Findlay & Pappin (1986) Biochem. J. 238, 625-642] suggests that the other disulphide bridge is formed between cysteine residues 110 and 187. A comparison is made of all the sequences of mammalian rhodopsins and colour pigments and attention is drawn to the fact that whereas Cys-322 and Cys-323 are conserved only in three rhodopsins (bovine, ovine and human), the residues corresponding to Cys-110 and Cys-187 are found in all the visual proteins (from rods as well as human cones).

| S-EPMC1148249 | biostudies-other

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