Oxidation-induced modification of the fibrinogen polypeptide chains

By using the mass-spectrometry method, the oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains induced by its oxidation have been studied. The αC-region has been proven to be the most vulnerable target for the oxidizer (ozone) as compared with the other structural elements of the Aα chain. The Bβ chain mapping shows that the oxidative sites are localized within all the structural elements of the chain in which the β-nodule exhibits high susceptibility to oxidation. The γ chains are the least vulnerable to the oxidizer action. The results obtained demonstrate convincingly that the self-assembly centers dealing with interactions of knob “A”: hole “a” are not involved in oxidative modification. It is concluded that the numerous oxidative sites revealed are mainly responsible for inhibiting lateral aggregation of protofibrils. The part of amino acid residues subjected to oxidation is supposed to carry out the antioxidant function.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Selective interaction of immunoglobulin polypeptide chains. Quantitative evaluation of the idiotypic and antigen binding activity of reassociated immunoglobulin molecules

We have analysed reassociated Ig molecules, containing heavy (H) or light (L) chains of Ig-B2 monoclonal antibody with human fibronectin binding activity and L of H chains of normal mouse serum immunoglobulin (Ig-NM). Examination of Ig-B2 idiotype expression in reassociated Ig indicated that 0.4% L-NM and 0.8% H-NM were able to restore Ig-B2 idiotype. The analysis of antigen binding capacity of reassociated Ig demonstrated, that only 4% H-NM created antigen binding site in complex with L-B2. We have determined the leading role of L-chain in creation of idiotype and binding site of Ig-B2. Selectivity of interaction between H and L chains is discussed. The results indicate, that not more than 4-6% of random H--L combinations produce functional Ig.     

Variation in the N-terminal sequence of heavy chains of immunoglobulin G from rabbits of different allotype

The sequences of the N-terminal peptides prepared by Pronase digestion of the heavy chain of rabbit immunoglobulin G of allotype Aa1, Aa2 and Aa3 were determined and were shown to be related to the allotype. An N-terminal fragment of about 34 residues was also prepared from the allotype heavy chains, by cleavage with cyanogen bromide; the yield varied with the allotype. The sequences of the cyanogen bromide fragments from the Aa1 and Aa3 heavy chains contain allotype-related variations similar to those found in the N-terminal Pronase peptides, and these sequences are thought to be representative of the whole heavy-chain populations. There is about 60% homology between the two sequences, and superimposed on the differences between them there are a number of positions within each sequence at which at least two amino acids are present.     

Optical anisotropy of polypeptide chains

Optical anisotropies γ² of N-t-butylacetamide (tBA), N-Methylacetamide (MA), and N, N-dimethylacetamide (DMA) have been determined from the Rayleigh ratios for depolarzed scattering by dilute solutions of the amides in p-dioxane. Traceless optical polarizability tensors \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for the amides are derived from these results in conjunction with the Kerr constant for tBA determined by LeGèvre and co-workers. It is shown that the tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}_i for the glycyle unit in a polypeptide chain may be identified with \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}MA . Methods for deriving corresponding tensors for other peptide units are indicated and the traceless polarizability tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for a polypeptide chain in any specified configuration is formulated.