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The disulphide bridges of a mouse immunoglobulin G1 protein.

ABSTRACT: [(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.

SUBMITTER: Svasti J

PROVIDER: S-EPMC1178493 | biostudies-other | 1972 Feb

REPOSITORIES: biostudies-other

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Interchain disulphide bridges of mouse immunoglobulin M.

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The disulphide bridges of immunoglobulin kappa-chains.

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| S-EPMC1270114 | biostudies-other

Disulphide bridges of the heavy chain of human immunoglobulin G2.

Project description:Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.

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Molecular basis for the dissociation dynamics of protein A-immunoglobulin G1 complex.

Project description:Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.

| S-EPMC3680412 | biostudies-literature

Disulphide bridges of bovine factor X.

Project description:Evidence is presented for the disulphide bridges in bovine Factor X. The protein was degraded by chemical and enzymic means, and all 12 disulphide bridges were isolated in separate peptides except for bridges nos. 6/7 in the light chain. All the disulphide bridges were found to be in positions corresponding to those found in other homologous domains. This report is the first verification of an epidermal-growth-factor-homologous domain having the same disulphide-bonding pattern as that found in mouse epidermal growth factor.

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Project description: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.

| S-EPMC1198754 | biostudies-other

Disulphide bridges of phospholipase C of Chlamydomonas reinhardtii modulates lipid interaction and dimer stability.

Project description:BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions.

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The disulphide bridges of bovine chymotrypsinogen B.

Project description:The five cystine peptides of chymotrypsinogen B have been isolated as pairs of cysteic acid peptides by the diagonal electrophoretic technique of Brown & Hartley (1963, 1966). The sequences of these peptides have been shown to be very similar to those of chymotrypsinogen A, and indicate that both zymogens have the same pattern of disulphide bridges. The determination of the sequence of residues 14-21 in chymotrypsinogen B has shown that residue 18 differs in the chymotrypsinogens from the corresponding residue in trypsinogen. Eight differences between chymotrypsinogens A and B are found in sequences accounting for 72 of the 245 residues.

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