Antibody organization on lipid films. Influence of pH and interchain disulphide reduction.
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ABSTRACT:
SUBMITTER: Uzgiris EE
PROVIDER: S-EPMC1149654 | biostudies-other | 1990 Nov
REPOSITORIES: biostudies-other
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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.
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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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