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W Palm

Publications and source records attributed to W Palm.

At least 19 recordsLinked to original sources
PubMed1989

[Proof of a disulfide bridge accessible to disulfide exchange between the heavy chains of IgG].

The paper deals with the direct experimental proof that human immunoglobulin G1 (IgG1) contains a reactive disulfide bond that can be opened by 3,3'-dithiobis(6-nitrobenzoate) (DTNB) within 24 h by a SH-catalysed disulfide exchange reaction. These results were obtained with the purified IgG1 myeloma protein and confirm earlier indirect evidence based on correlation analysis of DTNB reactivity and quantitative IgG1 determination. The reactive disulfide bond is most likely the one between Cys235 of the heavy chains in the "hinge"-region, activated for the disulfide exchange by the protonated amino groups of Lys231 as turned out by analysis of IgG1. As with the whole molecule, one mol of reactive disulfide was found per mol of the Fc-fragment. 0.8 mol of labile S-S bonds was detected per mol of F(ab)2. After separation of the excess of reagent, the sedimentation pattern still corresponded with the dimer. The unaltered antigenic properties as well as the crystallizability speak against any severe conformational changes. Therefrom it was concluded that in approximately 80% of the F(ab)2 molecules one of the two inter heavy chain-bridges was opened. With the isolated F(ab)-fragment a reaction with DTNB was ascertained to an extent of 20%, which is probably due to an altered stability of the heavy-light chain-SS-bridge. However, no influence on the sedimentation pattern was observed. The intrachainar disulfide bonds of neither the heavy nor the light chain reacted with DTNB to a measurable extent.

Disulfides↗
PubMed1989

[The primary structure of crystallizable monoclonal immunoglobulin IgG1 Kol. II. Amino acid sequence of the L-chain, gamma-type, subgroup I].

The immunoglobulin Kol was the first intact antibody molecule which was characterized by high-resolution X-ray crystallography. Furthermore the complete amino-acid sequence of the heavy (H)-chain is known. Here we report the complete amino-acid sequence of the light (L)-chain of the monoclonal immunoglobulin Kol (IgG1). The polypeptide has an Mr of 22,781, consists of 216 amino acids and due to its structure is of the lambda-type. With the characteristic amino acids threonine, asparagine, threonine, glycine and lysine in positions 101, 114, 116, 154, and 165, respectively the Kol L-chain is of the Mcg isotype. With the proteins Mcg, Mot, Bur, Loc and Mem six myeloma-derived amino-acid sequences of the same isotype are known. The amino-acid sequence of the N-terminal variable part is characteristic of subgroup 1. This contribution completes the primary structure of IgG1 Kol.

Alkylation↗
PubMed1987

Comparative studies on partial microsequence-analysis of Bence-Jones proteins.

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.

Amino Acid Sequence↗
PubMed1983

[Three-dimensional structure determination of antibodies. Primary structure of crystallized monoclonal immunoglobulin IgG1 KOL, I].

The crystallizable myeloma immunoglobulin IgG1 KOL [allotype Gm(-1,4), (gamma 1, gamma)2] which is well characterized in its three-dimensional structure by X-ray diffraction analysis of high resolution has been proved to be homogenous by polyacrylamide gel electrophoresis. The H- and L-chains were separated by gel filtration after complete reduction and carboxymethylation and were characterized by amino acid analysis, end group determination and polyacrylamide gel electrophoresis, respectively. The intact IgG1 KOL was cleaved by cyanogen bromide and all CNBr-fragments were isolated and characterized. The reduced and carboxymethylated H-chain was digested by trypsin and the tryptic hydrolysate was separated by ion-exchange chromatography. Using different procedures of rechromatography 35 out of 37 tryptic H-chain peptides could be isolated in sufficient amounts, the missing 2 peptides were produced by tryptic digestion of 2 CNBr-fragments. The amino acid sequences of all tryptic peptides were determined using a modified Edman degradation method after separation of the enzymatic cleavage products by high-performance liquid chromatography (HPLC). The complete primary structure of the VH-part of the H-chain was established by isolation and partial sequence determination of overlapping peptides obtained from cleavage of the intact H-chain by Staphylococcus aureus proteinase. The gamma 1-H-chain KOL comprises 455 amino acid residues and belongs to subgroup III. The switch from the variable to the constant part occurs at position 126/127, thus making VH-KOL one of the longest variable parts among the yet known immunoglobulin H-chains. This is due to the hypervariable region Hhv4 which is made up by 17 amino acid residues (4-9 residues more compared with other VH-parts). Within this region a so far not described additional intrapeptidal disulfide bridge could be localized (Cys 105-Cys 110) that creates a short loop with antiparallel running peptide strains in beta-pleated sheet conformation. Its role in the three-dimensional structure of the antigen-binding site of the IgG1 KOL molecule is discussed using the data obtained from X-ray diffraction analysis.

Amino Acid Sequence↗
PubMed1978

[Fractures and dislocations of the cervical spine caused by hanging (author's transl)].

In comparison to the pattern of lesions of the soft tissue of the neck and the CVC in case of suicidal hanging 17 postmortal fracture-dislocations of the neck are investigated. It could be demonstrated that on principle in case of hanging the dens-fracture can occur. With respect to the hangman's fracture (Wood-Jones) a differentiation into 3 types is proposed.

Adult↗
PubMed1978

Small-angle X-ray studies of a human immunoglobulin M.

The conformation of a Waldenström immunoglobulin M (IgM) with antibody-like activity for X-ray contrast media, based on 3-amino-2,4,6-triiodobenzoic acid, was studied by small-angle X-ray scattering. The radius of gyration was determined as 12.1 nm, the maximum distance 35 nm, the volume 1900 nm3. A flat star-shaped model was found to be equivalent in scattering. Aggregation of IgM molecules seems to take place as side-by-side combinations of single molecules, manifesting itself as a relatively large increase of the radius of gyration and unchanged thickness of the flat aggregates.

Humans↗
PubMed1977

Small-angle X-ray studies of the human immunoglobulin molecule Kol.

The conformation of the human immunoglobulin molecul Kol [IgG I, kappa2 gamma2, Gm(f)+] was studied by small-angle X-ray scattering in 0.15 M NaCl solution. The radius of gyration was found to be 5.84 +/- 0.04 nm, the volume 329 +/- 15 nm3 and the molecular weight 150 000 +/- 10 000. Information on the overall shape was obtained by comparing the experimental scattering curve with the calculated curves for various models. The models were obtained by arranging the models found for the Fab and Fc fragments of the same immunoglobulin molecule in a different manner. A model which fits all the date and the form of the experimental scattering curve is presented.

Female↗
PubMed1976

Crystallographic structure studies of an IgG molecule and an Fc fragment.

The crystal structures of a human IgG antibody molecule Kol and a human Fc fragment have been determined at 4-A and 3.4-A resolution respectively, by isomorphous replacement. The electron-density maps were interpreted in terms of immunoglobulin domains based on the Rei and McPC 603 models (Kol) and by model-building (Fc). The Fab parts of Kol have a different quaternary structure from that observed in isolated crystalline Fab fragments, there being no longitudinal V-C contact in Kol. The Fc part C terminal to the hinge is disordered in the Kol crystals. It is suggested that the Kol molecule is flexible in solution, whereas fragments are rigid. In the Fc fragment both CH3 and CH2 show the immunoglobulin fold. The CH3 dimer aggregates as CH1-CL while CH2 are widely separated from each other. The carbohydrate bound to Fc is in fixed position. From these structures a hypothetical liganded antibody molecule has been constructed, which is assumed to be rigid.

Antigen-Antibody Reactions↗
PubMed1976

Small-angle X-ray studies of the Fab and Fc fragments from the human immunoglobulin molecule Kol.

The conformation of the Fab and Fc fragments from the human immunoglobulin molecule Kol [IgI I, chi2gamma2, Gm(f)+] was studied by small-angle x-ray scattering in solution. The fragments were studied in 0.02 M Tris-HCl buffer. For the Fab fragment the radius of gyration was found to be 3.15 +/- 0.15 nm, the volume to be 75 +/- 8 nm3. For the Fc fragment the respective values were 3.15 +/- 0.15 nm for the radius of gyration and 91 +/- 8 nm3 for the volume. A large number of models were calculated for both fragments to find models which fit these data and have the same scattering curve. The models with the best agreement were compared with the models found for the crystalline state by crystal x-ray studies.

Humans↗
PubMed1976

[Physico-chemical characterisation and crystallisation of immunoglobulins and their fragments: a contribution to the three dimensional structural determination of antibodies, III. Human immunoglobulin Kol, a myeloma protein of the type IgG, gamma2 kappa2, Gm (f)+. Comparative studies on dissolved and crystalline material (author's transl)].

Immunoglobulin Kol has been isolated and characterised as the intact molecule, which consists of an antigen binding part (Fab) and the C-terminal fragment (Fc). Crystals of the protein are suitable for X-ray diffraction resolution of the fine structure, down to atomic dimensions; but this does not permit the representation of the Fc-part of the molecule. The following studies were performed on the protein in solution before and after crystallisation; the results were the same in both cases, thus showing that crystallisation does not alter the molecule: After enzymic cleavage of the protein, the products were isolated and identified by immunological reactions and by analytical ultracentrifugation; the behaviour of the protein in disc electrophoresis following reductive cleavage was also studied.

Chromatography, DEAE-Cellulose↗
PubMed1976

[Physico-chemical characterisation and crystallisation of immunoglobulins and their fragments: a contribution to the three-dimensional structural elucidation of antibodies, IV. The Fab fragment and the Fc fragment of human immunoglobulin Kol; isolation and crystallisation (author's transl)].

The X-ray fine structure analysis of intact crystalline immunoglobulin Kol indicates that the conformation of the Fab region differs essentially from that determined for the free Fab fragment. For this investigation, the free Fab fragment Kol was prepared and characterised, and crystalline preparations of the whole protein were obtained, that were suitable for an elucidation of atomic structure, and the appropriate measurements were made. In the crystal structure analysis of immunoglobulin Kol, it has not been possible to determine the Fc region. The Fc fragment was therefore separated from the whole molecule to facilitate its study. The isolation of this Fc fragment, its characterisation and subsequent crystallisation are described.

Chemical Phenomena↗
PubMed1975

The molecular structure of a dimer composed of the variable portions of the Bence-Jones protein REI refined at 2.0-A resolution.

The structure of the variable portions of a K-type Bence-Jones protein REI forming a dimer has been determined by X-ray diffraction to a resolution of 2.0 A. The structure has been refined using a constrained crystallographic refinement procedure. The final R value is 0.24 for 15000 significantly measured reflections; the estimated standard deviation of atomic positions is 0.09 A. A more objective assessment of the error in the atomic positions is possible by comparing the two independently refined monomers. The mean deviation of main-chain atoms of the two chains in internal segments in 0.22 A, of main-chain dihedral angles 6.3 degrees for these segments. The unrefined molecular structure of the VREI dimer has been published (Epp, O., Colman, P., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R., and Palm, W. (1974), Eur. J. Biochem. 45, 513). Now a detailed analysis is presented in terms of hydrogen bonds and conformational angles. Secondary structural elements (antiparallel beta structure, reverse turns) are defined. A more precise atomic arrangement of the amino acid residues forming the contact region and the hapten binding site is given as well as the localization of solvent molecules. Two cis-prolines (Pro-8 and Pro-95) were detected. The intrachain disulfide bridge (Cys-23-Cys-88) occurs statistically in two alternative conformations. The structure suggests reasons for strong conservation of several amino acid residues. The knowledge of the refined molecular structure enables crystal structure analyses of related molecules to be made by Patterson search techniques. The calculated phases based on the refined structure are much improved compared to isomorphous phases. Therefore the effects of hapten binding on the molecular structure can be analyzed by the difference Fourier technique with more reliability. Hapten binding studies have been started.

Amino Acid Sequence↗
PubMed1975

[The primary structure of a crystalline monoclonal immunoglobulin kappa-type L-chain, subgroup I (Bence-Jones protein Rei.); isolation and characterization of the tryptic peptides; the complete amino acid sequence of the protein; a contribution to the elucidation of the three-dimensional structure of antibodies, in particular their combining site (author's transl)].

Bence-Jones protein Rei. was isolated from the urine of a plasmacytoma patient by precipitation with ammonium sulfate and purified by ion exchange chromatography. The determination of the molecular weight showed that the protein was present in its monomeric form. The preparation cotained a minor fraction, which consisted of the dimer of the variable parts and was characterized by its ability to crystallize. X-ray data with a resolution on the atomic level could be achieved and allowed, together with the known primary structure, the construction of a three-dimensional model, which also gave an insight into the antigen binding site. It consists of the hypervariable regions, which form a pocket 15 A in diameter lying between the two monomers at the ends of the molecules. Sequence studies were performed with tryptic peptides which were purified by chromatographic procedures and aligned in homology to other kappa-chains. The protein belongs to subgroup I on the basis of its characteristic amino acid sequence and follows a highly regular pattern of amino acid exchanges. Its sequence is in agreement with an evolutionary origin of antibody variability.

Amino Acid Sequence↗