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N Hilschmann

Publications and source records attributed to N Hilschmann.

At least 19 recordsLinked to original sources
PubMed1992

Complete amino acid sequence determinations demonstrate identity of the urinary Bence Jones protein (BJP-DIA) and the amyloid fibril protein (AL-DIA) in a case of AL-amyloidosis.

The complete primary structures of both the main amyloid fibril protein component (AL-DIA) and the soluble Bence Jones protein (BJP-DIA) obtained from the same patient with AL-amyloidosis are reported for the first time. The amino acid sequences were determined by automated Edman degradation following proteolytic digestion of the isolated proteins and HPLC separation of the resulting fragments and by amino-terminal sequencing after treatment with pyroglutamate aminopeptidase. Sequencing data were confirmed by amino acid analysis and plasma desorption mass spectrometry (PDMS). Molecular weights of the complete proteins were determined by laser desorption mass spectrometry. The amyloid fibril preparation contained a complete monoclonal lambda immunoglobulin light chain (subgroup 1.2) as well as different-sized fragments thereof which were identified by immunoblotting and amino-terminal sequencing following immobilization of electrophoretically-separated proteins on poly(vinylidene difluoride) (PVDF) membranes. The soluble urinary Bence Jones protein (BJP-DIA) was a dimer of monoclonal L-chains with a primary structure identical to that of the amyloid L-chain (AL-DIA) and thus represented the amyloid precursor protein.

Aged↗
PubMed1992

Studies on human porin. VII. The channel properties of the human B-lymphocyte membrane-derived "Porin 31HL" are similar to those of mitochondrial porins.

Porin 31HL was isolated and purified from total membrane preparations of a human B-lymphocyte cell line. The protein showed a single band of apparent molecular mass 31 kDa on SDS-PAGE. Reconstitution of the protein into artificial lipid bilayer membranes defines its function as a channel-forming protein. The distribution of single-channel conductances had two maxima of 2.4 and 4.3 nS in 1M KCl. The channel formed by Porin 31HL of human B-lymphocytes was found to be voltage-dependent and switched to ion-permeable substates at membrane voltage larger than 20mV. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.7 nm. The properties of the low conductance state of the channel were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. Monoclonal antibodies against the N-terminal end of Porin 31HL blocked its reconstitution but had otherwise no influence on the channel properties. This result suggested that the amphipathic alpha-helical structure at the N-terminal end is probably not involved in channel gating. The channel-forming properties of Porin 31HL were compared to those of porins isolated from mitochondrial outer membranes and to those of the "maxi chloride channel" observed in the cytoplasmic membrane of several eukaryotic cells.

Animals↗
PubMed1992

Intra- and interchain disulfide bridges of the human J chain in secretory immunoglobulin A.

All intra J chain disulfide bridges of human sIgA, the disulfide bonds between the J chain and the two IgA monomers, and one inter IgA monomer disulfide bridge were determined. sIgA was isolated from colostrum of healthy women and digested with IgA1-specific protease followed by cyanogen bromide cleavage. This procedure generated fragments of 140 kDa, 60 kDa, and 28 kDa. The 28-kDa polypeptide comprised the complete J chain covalently bound to two alpha 1 chain octapeptides derived from the C-termini of two alpha 1 chains. The 28-kDa fragment was digested with trypsin. The resulting peptides were purified by RP-HPLC, and subsequently characterized by amino-acid analysis, mass spectrometry, and gas phase sequencing. These data unequivocally show that the J chain cysteines C1-C6, C4-C5, and C7-C8 form intra chain disulfide bridges. The second (C2) and the third (C3) J chain cysteines are disulfide linked to two alpha chain cysteines (C17) joining the two IgA monomers of sIgA tail to tail. The remaining two alpha chains of the two monomers are directly bound to each other via their ultimate cysteines (C17-C17). A new model for the J chain in sIgA is presented.

Amino Acid Sequence↗
PubMed1992

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibodies, Monoclonal↗
PubMed1991

[A flip-flop model of the chloride channel complex explains the dysregulation of the chloride flow in the plasmalemma of cells in cystic fibrosis].

The basic defect in cystic fibrosis is the chloride impermeability of the plasmalemma in different cells. A candidate for the chloride channel, thought to be affected in the syndrome, is "Porin 31HL" recently described by us. The molecule is i) expressed in the plasmalemma of different cells, it has ii) a molecular mass of 31,000 Daltons, it shows iii) high conductance in artificial membranes and it can be iv) modified by 4,4'-Diisothiocyanatostilbene-2,2'-disulfonate. A porin in the outer membrane of cells should furthermore v) be regulated by modulators. All these characters of "Porin 31HL" correspond to those given in literature for chloride channels. The regulation of the channels can be explained by a two component flip flop model.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Determination of the molecular structure of the human free secretory component.

Here, we present the experimental data, leading to determination of the primary structure, the linkage of the carbohydrates and the arrangements of the disulfide bonds of the human free secretory component. Methods of protein chemistry were used. The protein can be divided into five homology regions and is a member of the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1991

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha(2-8) polysialic acid. 1) Amino-acid sequence of the light (L-) chain, kappa-isotype.

Here we report the complete amino-acid sequence of the anti-alpha(2-8)polysialic acid antibody mAb735 light chain. The sequence was determined after digestion of the reduced and carboxymethylated L-chain with trypsin, SV-8 proteinase, and Asp-N proteinase, isolation of the generated peptides by RP-HPLC and characterization of these fragments by sequence analysis, amino-acid analysis and/or plasma desorption mass spectrometry. According to Kabat et al. the variable region belongs to the V kappa-II subgroup, whilst according to Hum et al. it belongs to the V kappa-1B subgroup. With the exception of proline at position 46, the sequence from position 1 to 95 is identical to the translated DNA sequence of a V kappa-germline gene segment previously reported.

Amino Acid Sequence↗
PubMed1991

Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC "Porin 31HL" and their application for histotopological studies in human skeletal muscle.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL. In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC "Porin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin. Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines. VDAC in the plasmalemma is discussed as the molecular basis of the Blatz and Magleby channel.

Animals↗
PubMed1991

Studies on human porin. IV. The primary structures of "Porin 31HM" purified from human skeletal muscle membranes and of "Porin 31HL" derived from human B lymphocyte membranes are identical.

We report on the purification of "Porin 31HM" from the crude plasma membrane fraction of human skeletal muscle. Furthermore, all tryptic peptides of the molecule were purified and characterized by different methods. The alignment of the peptides with the complete primary structure of the human B lymphocyte plasma membrane-derived "Porin 31HL", published by us recently (Kayser, H. et al. (1989) this Journal 370, 1265-1278), proved both structures to be completely identical. Our data demonstrate that porin fractions from crude plasma membranes of different human cell types do not show any variation on the primary structure level.

Amino Acid Sequence↗
PubMed1991

The complete amino-acid sequence of the heavy chain of the human myeloma protein WIE, an immunoglobulin D.

The human myeloma protein WIE is a lambda-type immunoglobulin D; the amino-acid sequence of its Fc part and aminoethylated heavy chain was completely determined. The VH-part (subgroup III) begins N-terminally with 5-oxoproline, and it contains a long, unique CDR3 region. Since the constant part differs from known delta chains by one amino-acid substitution in the hinge region, IgD WIE probably represents an allotypic variant. As in other protein delta chains, O-glycosylations are confined to the hinge region. Furthermore, the ratios of N-glycosylations at the three positions are identical in IgD WAH [Takahashi, N. et al. (1984) J. Chromatogr. 317, 11-26.] and IgD WIE (100%, 50%, 100%). From the most conserved constant domain, C delta 3, a three-dimensional model was constructed to clarify the role of its delta-specific substitutions and glycosylation.

Amino Acid Sequence↗
PubMed1991

[Studies on human porin. V. The expression of "porin31HL" in the plasmalemma is not by cell transformation].

In recent papers we proved "Porin 31HL" to be located on the surface of human, EBV-transformed B lymphocytes. Here we present proof of "Porin 31HL" in the plasmalemma of normal human blood lymphocytes. For this purpose B and T lymphocytes were isolated from human heparinized blood and examined by indirect immunofluorescence techniques using different monoclonal antibodies against purified "Porin 31HL" and some B and T cell markers, respectively. For comparison a number of established cell lines of different origin were employed. Hence it followed that normal B and T cells as well as transformed and leukemic cells express "Porin 31HL" in their membrane. No significant quantitative differences could be seen. Consequently, the location of "Porin 31HL" in the plasmalemma is not a product of transformation.

Animals↗
PubMed1990

Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?

"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1989

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers.

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.

Amino Acid Sequence↗
PubMed1989

[Identification of human porins. I. Purification of a porin from human B-lymphocytes (Porin 31HL) and the topochemical proof of its expression on the plasmalemma of the progenitor cell].

We describe for the first time a porin (Porin 31HL) on the plasmalemm of an eukaryontic cell line, where porins have been found only on the outer mitochondrial membranes. The expression of the porin on the plasmalemm of transformed human B-lymphocytes is demonstrated by cytotoxicity- and indirect immunofluorescence techniques with living and fixed cells. The rabbit xenoantisera used were directed against purified Porin 31HL and free or acetylated synthetic peptides of its nineteen N-terminal amino acids. The three-step purification procedure for Porin 31HL started from a total membrane fraction of the B-cell line, followed by ion-exchange chromatography on CM- and DEAE-cellulose and a final gel filtration in SDS on Sephacryl S-300.

Amino Acids↗
PubMed1989

[Identification of human porins. II. Characterization and primary structure of a 31-lDa porin from human B lymphocytes (Porin 31HL)].

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.

Amino Acid Sequence↗
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

Structural relationship of an apolipoprotein (a) phenotype (570 kDa) to plasminogen: homologous kringle domains are linked by carbohydrate-rich regions.

At least six allelic forms of apolipoprotein(a), differing in molecular mass, could be detected by immunoblot analysis. One of these phenotypes with a molecular mass of 570 kDa has been investigated. After reduction and carboxymethylation it was digested with trypsin and the resulting peptides were separated by gel filtration and reverse phase HPLC. The tryptic fragments sequenced comprised a total of 356 amino acids. The N-terminus of apo(a) was highly homologous to the start of the kringle 4 domain from human plasminogen and the majority of the tryptic peptides isolated was also homologous to sequences from this kringle. At least five homologous "kringle 4" domains are present in apolipoprotein(a) whereby one domain occurs more frequently than the others. A carbohydrate-rich peptide was also obtained in high yield. This glycopeptide connects two "kringle 4" domains and contains one N-glycoside within the kringle and six potential O-glycosides in the linking region. From the recovery it can be estimated that this peptide occurs several times within the whole apolipoprotein (a) sequence. The high carbohydrate content is in sharp contrast to that of human plasminogen. Other peptides sequenced indicate that apo (a) also contains domains homologous to the kringle 5 and protease regions of plasminogen. No unique peptides were found. These studies suggest that apolipoprotein (a) could have arisen through duplication of specific regions from the human plasminogen gene. The size heterogeneity of apo (a) might then be explained by differences in the numbers of gene duplications.

Amino Acid Sequence↗