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Biomedical subjects

D G Braun

Publications and source records attributed to D G Braun.

At least 55 records · Page 3Linked to original sources
PubMed1983

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.

Allergens↗
PubMed1983

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.

Amino Acid Sequence↗
PubMed1983

Sphingosine determination at the picomole level using dimethylaminoazobenzene sulphonyl chloride.

A sensitive method (lower detection limit 5 picomoles) is proposed for the determination of sphingoid bases liberated from mammalian glycosphingolipids by acid hydrolysis. The azo dye 4-dimethylaminoazobenzene-4'-sulfonyl chloride reacts with the primary amino group of the sphingosine bases, forming a stable derivative. Excess reagent, which is degraded during the derivatization, and free amino sugars as common hydrolysis products of glycosphingolipids are completely separated by reversed-phase high-performance liquid chromatography. This method was applied to the determination of the glycosphingolipid content of mouse spleen and thymus.

Animals↗
PubMed1983

The complete amino acid sequence of the light chain variable region of two monoclonal anti-p-azobenzene-arsonate antibodies bearing the cross-reactive idiotype.

Two monoclonal anti-p-azobenzene-arsonate antibodies produced by cell fusion of A/J spleen lymphocytes were selected. It had previously been shown that they both expressed the cross-reactive idiotype (CRI) and that the amino terminal sequence of their heavy chain variable region differed by only one amino acid substitution within the first 55 residues, at residue 41 in the second framework region. A novel, sensitive peptide-mapping method had indicated that the light chain of these two antibodies differed by at least three amino acid substitutions. Here, the complete amino acid sequence of the light chain variable region is presented. There are only 3 amino acid substitutions between the two antibodies, located in the first and second complementarity determining regions and the third framework region respectively. Although the variable region of the light chains of these two monoclonal antibodies show such a high degree of homology they differ by 26 and 27 substitutions from the reference sequence of the light chain of CRI+ anti-ABA serum antibodies. In addition, they are homologous to 4 other such CRI+ monoclonal antibody light chain sequences published so far, in which only 2 of the above 3 substitutions are not represented. The contribution of the light chain to the CRI is discussed.

Amino Acid Sequence↗
PubMed1983

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes.

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.

Amino Acid Sequence↗
PubMed1983

Specificities of monoclonal antibodies against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis.

Eight hybrid cell lines secreting monoclonal antibodies directed against the activated delta-endotoxin of Bacillus thuringiensis var. thuringiensis were grown in BALB/c mice. Ascites fluids were collected, and the antibodies were purified by antigen-affinity chromatography. The specificity of each monoclonal antibody for the toxin and protoxin was established by the indirect enzyme-linked immunosorbent assay. All the antibodies consisted of gamma 1 heavy and kappa light chains. They were reactive with both the native toxin and the protoxin. In contrast to specific goat antiserum, they failed, however, to bind to heat and sodium dodecyl sulfate denatured antigen. These eight cloned cell lines gave rise to five kinds of antibodies distinguished by isoelectric focusing. Competitive antibody binding studies revealed that these five antibodies recognize at least four distinct antigenic determinants of the native toxin and the protoxin. Two of the epitopes are unrelated, whereas three antibodies compete for binding to their antigenic determinants. In the bioassay with larvae of Pieris brassicae, one antibody was found to block the toxin and protoxin activity completely. A second inhibited it partially, whereas the other three antibodies did not affect it at all.

Antibodies, Bacterial↗
PubMed1982

A complete separation of dimethylaminoazobenzenesulphonyl-amino acids. Amino acid analysis with low nanogram amounts of polypeptide with dimethylaminoazobenzenesulphonyl chloride.

A chromatographical system has been developed to give a complete baseline separation of all dimethylaminoazobenzenesulphonyl (DABS)-amino acids for high-sensitivity amino acid analysis [Chang, Knecht & Braun (1981) Biochem. J. 199, 547--556]. The system, which uses a Merck RP-18 column with phosphate buffer (12 mM, pH 6.5)/acetonitrile mixture, allows reliable analysis of DABS-amino acids at the 1--2 pmol level. The accuracy of this new system is demonstrated by the composition analysis of two immunoglobulin light chains (214 amino acid residues) with differences at only three amino acid residue positions.

Amino Acids↗
PubMed1982

Idiotypic and structural analysis of monoclonal human immunoglobulins with anti-streptolysin O activity.

The class, subclass and light chain distribution of eight myeloma proteins with anti-streptolysin O activity (ASO) in sera studied by our group and in cases from the literature indicates a predominance of the IgG1 kappa subclass and type of immunoglobulins. Three IgG myeloma proteins (O.I., L.F. and A.S.) were further characterized with respect to idiotypic cross-reaction and variable region subgroup. The anti-idiotypic antibodies (anti-Id) against protein O.I. and against protein L.F. both indicated idiotypic cross-reaction between O.I. and L.F. While in the case of anti-IdL.F. this cross-reaction was in the order of 5% it was much weaker when studied with anti-IdO.I.. The reaction between O.I. and anti-IdO.I. could be inhibited to 95% by the antigen streptolysin O, indicating that the idiotypic determinants are associated with the combining site. N-terminal amino acid sequence analysis showed that all three light chains fall in the V kappa III variable region subgroup. The heavy chains of proteins O.I. and L.F. with idiotypic similarities both have blocked N-termini, indicating the presence of the VHI or VHII subgroup while protein A.S. belongs to the VHIII subgroup.

Amino Acid Sequence↗
PubMed1982

Murine VK25 isotype sequence: monoclonal antibody 2S1.3 specific for the group A streptococcal polysaccharide.

Antibodies 2S1.3 and 2S1.1 are hybridoma derived gamma 3K murine immunoglobulins with specificity for N-acetylglucosamine beta 1 leads to 3-linked to L-rhamnose, the immunodeterminant of the streptococcal group A polysaccharide. The VL 2S1.3 amino acid sequence is the second complete one reported with this specificity, and it is the first fully determined VK25 structure. The VK regions of this and the previously described monoclonal anti-A-CHO antibody 7S34.1 are compared with 17 selected partial and complete murine VK regions, preferentially from anti-polysaccharide antibodies. Both VK 2S1.3 and 7S34.1 increase the variability of existing murine VK regions; however, they are, as anti-A-CHO antibodies, most homologous to each other sharing the so far unique residue argine 74. The amino acid sequence of the constant region of 2S1.3 light chain was also determined. It was found to be identical with that of MOPC21, but different from MOPC41 and MOPC70 in residue positions 127, 129, 163 and 166.

Amino Acid Sequence↗
PubMed1982

A sensitive peptide mapping method. Identification of three amino acid substitutions within two anti-azobenzenearsonate monoclonal antibody light chains.

Peptide mixtures, precolumn-derivatized with dimethylaminoazobenzene isothiocyanate, have been separated by reversed-phase high-performance liquid chromatography to generate a dimethylaminoazobenzene thiocarbamoyl peptide map. The eluted peptide derivatives are detected in the visible region with a sensitivity of 2-5 pmol and can be collected for direct structural analysis. This technique was applied to compare the sequence homology of two immunoglobulin light chains which were derived from two anti-azobenzenearsonate monclonal antibodies, namely 10K44-7A1 and 10K26-12A1. The complete variable region sequences of 10K44-7A1 and 10K26-12A1 light chains were established based on the sequence analysis of tryptic peptides, intact light chains and reference sequences obtained previously [Siegelman M. and Capra, J.D. (1981) Proc. Natl Acad. Sci. USA, 78, 7679-7683]. Altogether, three amino acid substitutions have been detected within complementary determining regions 1 and 2, and framework region 3, all requiring only a single base change at the DNA level. This new technique provides detection limits and the feasibility of analysing peptides which are not obtainable with conventional techniques.

Amino Acid Sequence↗
PubMed1982

Ganglioside patterns: new biochemical markers for human hematopoietic cell lines.

The ganglioside patterns from a representative collection of 20 human B-cell, T-cell, null cell, myeloid cell, and early erythroid cell lines were determined. Radioactive sugar-labeled gangliosides from cultured cells were extracted, analyzed, and quantitated by high-performance thin-layer chromatography (HPTLC) and high-pressure liquid chromatography, essentially according to the polarity of the oligosaccharide chain. The ganglioside labeling patterns of the human cell lines were in some cases extremely complex, since more than 100 components could be separated upon two-dimensional HPTLC, e.g., in the case of a promyelocytic cell line. Each type of cell showed a characteristic pattern: Null cell lines were much less complex than T-cell lines, and myeloid and erythroid lines exhibited characteristic patterns as well. Although clearly distinguishable from all other cell lines, the B-cell lines showed a high degree of heterogeneity. Therefore, gangliosides can be expected to serve as additional markers to establish the origin, of a cell line or to classify pathologic cell populations.

Antigens, Surface↗
PubMed1981

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.

Amino Acids↗
PubMed1981

Epstein-Barr virus strain- and group-specific antigenic determinants detected by monoclonal antibodies.

In order to differentiate Epstein-Barr virus (EBV) strains by serological markers we prepared hybridomas producing monoclonal antibodies against polypeptides of the QIMR-WIL EBV strain. These monoclonal antibodies were screened by indirect immunofluorescence techniques. Immunoprecipitation using EBV-positive monoclonal antibodies and the protein A method, of 125I-labelled polypeptides from purified QIMR-WIL EBV particles, revealed that a series of monoclonal antibodies against the four main surface and envelope polypeptides precipitating p340, p340/p240, p140, and p80 were obtained. When 125I-labelled polypeptides from purified P3HR-1 and B95-8 EBV particles were immunoprecipitated, it could be demonstrated that several anti-p340 (QIMR-WIL) antibodies recognized strain-specific antigenic determinants, while anti-p340/p240 (QIMR-WIL) as well as anti-p140 (QIMR-WIL) antibody clones reacted with antigenic sites which are in common wither among B95-8 or in addition to P3HR-1 polypeptides. Thus, monoclonal antibodies now provide a serological basis for EBV typing.

Animals↗