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N Koch

Publications and source records attributed to N Koch.

85 records · Page 5Linked to original sources
PubMed1983

Association of surface IgM with two membrane proteins on murine B lymphocytes detected by chemical crosslinking.

Surface proteins of intact murine B lymphocytes were crosslinked by the bifunctional reagent, 4,4'-diphenyldiazoniumdisulphidfluoroborate and then radiolabelled with 125I. After solubilization of the cells, Ig-containing complexes (Ig and protein(s) covalently crosslinked to Ig) were isolated and analyzed by two-dimensional SDS polyacrylamide gel electrophoresis (2D-SDS-PAGE). Ig-containing complexes were separated in the first dimension by cylindrical SDS gels. Subsequently, the disulphide bridges of the isolated surface Ig molecules and of the crosslinking reagent were cleaved, and the products electrophoresed in the second dimension on SDS slab gels. In addition to the Ig polypeptide chains, two proteins with mol. wts of 46,000 and 56,000 could be identified. In order to answer the question whether these proteins are associated with IgM and/or IgD Ig-complexes were separated with regard to their isotype and analyzed separately by 2D-SDS-PAGE. It was found that both proteins are associated with subunits of IgM. In the case of IgD, no associated structures could be demonstrated by the method used.

Animals↗
PubMed1983

Comparison of allogeneic and xenogeneic determinants on the H-2Kk molecule.

Two monoclonal rat anti-H-2 antibodies were derived by cell fusion from a rat immunized with mouse CH-1 lymphoma cells. Both antibodies recognize polymorphic H-2 determinants expressed on most haplotypes. Competitive binding studies on H-2Kk show that one rat antibody, R1-9.6, detects a determinant that is distant from the allodeterminants defined by 12 different mouse monoclonal anti H-2 antibodies. In contrast, the rat antibody R1-21.2 inhibits the binding of most mouse monoclonal anti H-2 antibodies to H-2k cells, possibly by causing a conformational change in the H-2 molecule. Both antibodies bind strongly to H-2 molecules and can be used for structural and biochemical studies.

Animals↗
PubMed1982

Cross-blocking studies with monoclonal antibodies against I-A molecules of haplotypes b, d and k.

For assessment of the distribution of allodeterminants on I-A molecules, binding inhibition studies were performed with monoclonal anti-I-A antibodies (mAb) in which pairs of labeled and unlabeled mAb were tested for cross-blocking on spleen cells of H-2 haplotypes b, d and k. The data suggest that allodeterminants are randomly distributed on the surface of the I-A molecule. Comparison of mAb with cross-reactivity for b, d or k haplotypes indicates that the allelic forms of determinants are located in analogous positions on different I-A antigens.

Alleles↗
PubMed1982

Subunits of IgM and IgD on the surface of murine B-lymphocytes.

Immunoglobulin isolated from 125I-labelled cell surface proteins of murine B-lymphocytes was analyzed by a sensitive two-dimensional polyacrylamide gel electrophoresis technique (2D-SDS-PAGE). Uncleaved Ig molecules were electrophoresed in the first dimension in an SDS-polyacrylamide gel, and after subsequent reduction of the disulphide bonds by mercaptoethanol, the cleaved polypeptides were separated in the second dimension using again an SDS-polyacrylamide gel. This technique enables the identification of unreduced Ig molecules and their corresponding subunit components. In the case of IgM as well as IgD the four chain structure (H2L2), half molecules (HL), and disulphide-linked heavy chains (HH) could be identified. Since all Ig subunits were isolated by an anti-IgG antiserum by virtue of its L-chain specificity we conclude that L-chains are noncovalently associated with the disulphide-linked heavy chains (HH). Free noncovalently bound L-chains could actually be identified by 2D-SDS-PAGE. However, this technique does not determine whether half Ig molecules (HL) are noncovalently associated with each other. In addition to free L-chains noncovalently linked mu and delta-chains were found. Control experiments showed that the identified Ig subunits are not artefacts of the isolation procedure or reduction moieties of H2L2 molecules (e.g. incubation of isolated mu 2L2 and delta 2L2 with detergent extracts of spleen cells does not result in the formation of subunits). On the basis of 125I-radioactivity incorporated into the Ig subunits it was estimated that besides mu 2L2 and delta 2L2, delta L (40-50% of total IgD) is the main Ig structure on B-lymphocytes. The other Ig subunits (mu L, delta 2, structures (ca. 10(4) molecules/B-cell) are sufficient to act as antigen recognition structures and to be involved in B-lymphocyte triggering and tolerance induction.

Animals↗
PubMed1982

Structure of Ia antigens: identification of dimeric complexes formed by the invariant chain.

A 2-dimensional gel technique was used for the identification of disulfide-linked polypeptide complexes formed by Ia chains and their corresponding subunits. Several dimers containing the invariant (Ii) chain were found. The most prominent one consisted of 2 Ii chains. In addition, Ii molecules were found to be covalently linked to 3 unknown components of 41,000, 27,000, and 10,000 to 15,000 m.w. The dimeric form of Ii exists also after alkylation of free SH-groups with iodoacetamide, which suggests that formation of the Ii dimer is not the result of experimental conditions. Furthermore, noncovalently associated dimers formed by processed alpha- and beta-chains as well as by their precursors alpha p and beta p could be detected. No mixed associated chains composed of precursors and processor molecules were observed, which indicates that processing and insertion into the membrane take place only after dimerization.

Alkylation↗
PubMed1981

Ia antigens contain two distinct forms of beta chain.

Two new forms of beta chain, beta 1, and beta 2, in I-A immunoprecipitates are described, which differ in their migration values in SDS-PAGE under nonreducing conditions, but which migrate identically in a reduced form. This behavior is very likely due to a different arrangement of intramolecular disulfide bonds which may influence mobility in SDS-PAGE. Peptide map analysis confirmed that beta 1 and beta 2 possess identical primary polypeptide structures. These two forms of beta chain are also expressed on the cell surface and its is suggested that both associate with alpha chains. The structural differences in these complexes may lead to an increase in heterogeneity of Ia antigens which could be of importance for T-cell recognition.

Animals↗
PubMed1981

Radioiodination of surface proteins and glycoproteins of lymphocytes by immobilized lactoperoxidase.

Radioiodination of lymphocyte surface proteins employing soluble lactoperoxidase was found to be unsatisfactory for the quantitative isolation and characterization of cell surface glycoproteins: either the glycoproteins were contaminated by self-iodinated lactoperoxidase or were in part removed by the washing step following the radioiodination procedure. Therefore, optimal conditions for cell surface radioiodination by lactoperoxidase covalently linked to Sepharose 4B (lacto-beads) were worked out. By the employment of immobilized lactoperoxidase, the enzyme could easily be removed following solubilization of the radiolabelled cells by a simple washing step. Radioiodinated cell surface proteins and glycoproteins were obtained without any loss as shown by SDS polyacrylamide gel electrophoresis.

Animals↗
PubMed1980

The detection of associated erythrocyte membrane proteins by cross-linking and surface radiolabelling.

Membrane proteins of intact erythrocytes were cross-linked using the cleavable homobifunctional reagent, 3,3'-dithiobis(propionimidate) and the non-cleavable heterobifunctional reagent, 4-azidobenzimidate. Subsequently, the exposed cross-linked membrane proteins were radiolabelled by the lactoperoxidase method. Cross-linked and radiolabelled membrane proteins were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis in which resolution in the second dimension was preceded by mercaptoethanol treatment. Complexes so formed by 3,3'-dithiobis(propionimidate) were cleavable, whilst components cross-linked by 4-azidobenzimidate were uncleavable. Three complexes were found. Two of these contained protein III as the only radiolabelled component. It was assumed that the first (Mr approx. 200000) represents a dimer and the second (Mr approx. 300000) an oligomer of protein III. The third complex, which was identified after cross-linking with 4-azidobenzimidate, consisted of PAS II and a low molecular weight (lipid-like) component. Since PAS I (dimer of a PAS II) was not associated with this component it was concluded that PAS I and PAS II are situated in different environments of the erythrocyte membrane.

Autoradiography↗
PubMed1975

Melanoma cells resistant to inhibition of growth by melanocyte stimulating hormone.

Melanocyte stimulating hormone (MSH) enhances melanization but inhibits proliferation of Cloudman S91 melanoma cells in culture. We have isolated variants of these cells that can grow in the presence of MSH. The conclusions we have reached from analyses of these cells are the following: (1) Basal tyrosinase activity (monophenol monooxygenase; monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), i.e., the activity that is present in the absence of added MSH, is related through a common biochemical pathway to MSH-mediated control of growth. (2) MSH-inducible tyrosinase activity does not appear to be related to MSH control of growth. (3) The morphological changes that occur following the addition of MSH or cAMP are related to controls of growth and not to those of melanization.

Animals↗