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W Voelter

Publications and source records attributed to W Voelter.

At least 91 records · Page 5Linked to original sources
PubMed1998

Purification and primary structure of low molecular mass peptides from scorpion (Buthus sindicus) venom.

The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35-36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55-78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40-80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.

Animals↗
PubMed1998

Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity.

This report demonstrates that in vitro activation of human cells with the beta-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK- and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and IL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.

CD4-Positive T-Lymphocytes↗
PubMed1997

Complete amino acid sequence of dioxygen-binding functional unit of the Rapana thomasiana hemocyanin.

The complete amino acid sequence of the Rapana thomasiana hemocyanin N-terminal functional unit Rta was determined by direct sequencing and matrix-assisted laser desorption ionization mass spectrometry of the protein and peptides obtained by cleavage with EndoLysC proteinase, TPCK-trypsin and cyanogen bromide. The single polypeptide chain consists of 407 residues. This is the first report on the primary structure of a dioxygen-binding unit from a marine gastropod hemocyanin and of an N-terminal domain from a molluscan dioxygen carrier. Comparison with the sequences of other molluscan hemocyanin functional units shows an average identity of 48 +/- 5 %. Inspection of the Rta sequence revealed residues 27 and 250 as carbohydrate attachment sites. Conclusions about the molecular evolution of the molluscan hemocyanin dioxygen-binding functional units are made.

Amino Acid Sequence↗
PubMed1997

Capillary electrophoresis in clinical chemistry.

Since its introduction, capillary electrophoresis has diversified, spreading out into different specialized fields covering solutions for almost any analytical questions arising in research laboratories. In the context of clinical chemistry, results must be provided at low costs and in a clinically relevant time frame; however, the attributes which have made capillary electrophoresis such a successful tool in basic research are identical to those attracting clinical laboratories: speed (more efficient, less labor-intensive), low costs (minimal buffer consumption), small sample volume (reduced blood collection volume from patient), increased selectivity (determination of multiple solutes in one run), and versatility (detection of analytes over the wide range of molecular masses and chemical composition). Nevertheless, it should be mentioned that there are still some drawbacks at this stage to be solved in the near future, such as lack of sensitivity for many clinical applications or the constraint to measure in a sequential mode. The aim of this survey is to familiarize clinical chemists, as well as chemists, with a short introduction to capillary electrophoresis, followed by chapters reviewing prominent fields of applications and the latest developments in clinical chemistry.

Body Fluids↗
PubMed1997

Conformation of thymosin beta 9 in water/fluoroalcohol solution determined by NMR spectroscopy.

The conformation of thymosin beta 9 in solution of 40% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol-d2 in water has been investigated by two-dimensional 1H-nmr spectroscopy. Under this condition thymosin beta 9 adopts an ordered structure. The determination of the conformation of the peptide was based on a set of 304 approximate interproton distance constraints derived from nuclear Overhauser enhancement measurements. The conformation of thymosin beta 9 includes two helical regions from residues 4 to 27 and 32 to 41. The two helices are separated by a poorly defined loop region between amino acids 28 and 31; the N-terminus of thymosin beta 9 shows random-coil structure only.

Amino Acid Sequence↗
PubMed1997

Prothymosin alpha: efficient sequence determination by experimental and theoretical capillary electrophoretic support.

A rapid and efficient primary structure elucidation of prothymosin (ProT alpha) and its enzymatic fragments was performed by a combination of matrix-assisted laser desorption ionization mass spectrometry (MALDI), automatic N-terminal Edman degradation, and the available theoretical predictions of electrophoretic mobility in capillary electrophoresis (CE) as a basis suggesting active sites of ProT alpha in the different bioassays.

Amino Acid Sequence↗
PubMed1997

Amino-terminal oxygen-binding functional unit of the Rapana thomasiana grosse (gastropod) hemocyanin: carbohydrate content, monosaccharide composition and amino acid sequence studies.

The amino-terminal oxygen-binding unit Rta of the Rapana thomasiana hemocyanin is a glycoprotein with a carbohydrate content of 4.8% (w/w). Sugar analysis revealed as monosaccharide constituents xylose, fucose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. On subtracting the carbohydrate contribution from the molecular mass of 49,698 Da, determined by laser desorption mass spectrometry for Rta, an M(r) value of 47,318 Da was determined for the polypeptide part of the functional unit. The Rapana hemocyanin oxygen-binding unit Rta contains 400 residues in a single polypeptide chain. The nearly complete amino acid sequence (about 90%) is determined. This is the first report on a sequence of a marine gastropod oxygen-binding unit and also on a molluscan hemocyanin amino-terminal unit. Comparison of the Rta sequence with those of other molluscan hemocyanin units, localized in the C-terminus or in the middle of the respective multidomain polypeptide chains, revealed 42-46% homology (52-55%, including isofunctional residues). Probably, all molluscan oxygen-binding units evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1996

Complete amino acid sequence of the A chain of mistletoe lectin I.

The complete amino acid sequence of the A chain of mistletoe lectin I was determined via Edman degradation sequencing of the N-terminus and tryptic and endoproteinase Asp-N overlapping fragments, amino acid analysis and MALDI-MS. The data obtained show a great homology with the chains of ribosome-inactivating proteins such as ricin and abrin with 111 (abrin-a) and 103 (ricin-D) amino acid residues conserved, respectively. The knowledge of the primary structure of MLA will have a fundamental impact on elucidating the biological function of medically applied mistletoe lectins on a molecular basis.

Amino Acid Sequence↗
PubMed1996

Screening and identification of familial defective apolipoprotein B-100 in clinical samples by capillary gel electrophoresis.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder. It is characterized by a decreased affinity of low density lipoprotein (LDL) for the LDL receptor, as a consequence of a substitution of adenine by guanine in exon 26 of the apolipoprotein B-100 gene, coding for the putative LDL receptor-binding domain of the mature protein. This disorder is associated with a strikingly high incidence of arteriosclerosis and tends to cause disease and premature death. In this communication we describe a rapid capillary gel electrophoretic method in combination with molecular biology techniques to facilitate the diagnosis of FDB. Mutation screening for FDB is performed by an allele-specific amplification followed by capillary gel electrophoresis (CGE). For the combined polymerase chain reaction (PCR)-CGE method, a total analysis time of only 3 h is needed, a period that is normally necessary for the run and for staining of the gel only, not including the time for PCR, gel casting, etc. In our pilot study 4 of 43 hypercholesterolemic patients were found to have the predominant apoB 3500 codon mutation. The verification is demonstrated by DNA-sequencing. This pilot study will be followed by a large cohort analysis of the south-west German population to determine the frequency of FDB in this area. The PCR-CGE method on the Dionex capillary electrophoresis system (CES I) allows rapid, fully automated detection of the mutation resulting in the unequivocal diagnosis of FDB.

Adult↗
PubMed1996

The ternary complex of DNase I, actin and thymosin beta4.

We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.

Actins↗
PubMed1996

Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits.

1. The stability towards pH changes, thermal and chemical (guanidine hydrochloride) denaturation of the oxy- and apo-forms of the native Rapana thomasiana haemocyanin and its structural subunits, RHSS1 and RHSS2, has been investigated using fluorescence and CD spectroscopy. The association of the subunits into haemocyanin aggregates increases considerably the melting temperature and the free energy of stabilization in water. The guanidine hydrochloride denaturation of the aggregated oxygen-transporting protein depends slightly on the protein concentration. The denaturation of the individual subunits is concentration-independent. Rapana haemocyanin is 5.9-7.5 kJ/mol more stable than the constituent polypeptide chains. 2. Upon excitation of the native haemocyanin and the subunits at 295 or 280 nm the fluorescence emission is determined by tryptophyl residues 'buried' deeply in the hydrophobic interior of the protein globules. This is confirmed by quenching experiments with acrylamide, caesium and iodide ions. The efficiency of the radiationless energy transfer between the phenol (donor) and indole (acceptor) fluorophores in the three species, native haemocyanin, RHSS1 and RHSS2, has been determined. An efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs in the non-dissociated form of the haemocyanin. 3. The tryptophan emission of the oxyhaemocyanin, oxy-RHSS1 and oxy-RHSS 2 is strongly quenched by the copper-dioxygen complex at the active site and the respective quantum yields of fluorescence of the oxygenated species are 4-7 times lower than those of the apo-forms. Protonated imidazole groups quench the fluorescence of neighbouring exited indole rings, probably by charge-transfer complex formation.

Animals↗
PubMed1996

Hydroxyalkenal formation induced by advanced glycosylation of low density lipoprotein.

Advanced glycosylation end products (AGEs) have been identified to be present on both the apolipoprotein and lipid components of low density lipoprotein (LDL) and to act to prevent its recognition and uptake by high affinity, tissue LDL receptors. Lipid-linked AGEs form readily in vitro by the covalent addition of glucose to the amine-containing head groups of phospholipids. This process is accompanied by oxidation of the unsaturated fatty acid side chains and occurs in the absence of exogenously added transition metals or free radical generating systems, suggesting that AGE formation may contribute significantly to lipid oxidation in vivo. To assess more precisely the chemical basis of AGE-induced oxidative modification, we performed gas chromatography-mass spectrometry analysis of the lipid products which form over time during LDL-advanced glycosylation in vitro. Negative ion chemical ionization mass spectroscopy of two major compounds that were identified were consistent with the structures of the fatty acid oxidation products 4-hydroxyhexenal and 4-hydroxynonenal. These data support the concept that AGE formation in close proximity to unsaturated fatty acyl groups leads to lipid oxidation and provide additional evidence that advanced glycosylation is an important pathogenic modification of the LDL particle in vivo.

Aldehydes↗
PubMed1996

Identification of contact sites in the actin-thymosin beta 4 complex by distance-dependent thiol cross-linking.

Binding sites of actin and thymosin beta 4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys10, Cys374, or the sulfur atom of the ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S), the actin derivatives were reacted with synthetic thymosin beta 4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys374 of actin and Cys6 of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (< or = 9.2 A) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta 4 analogs when the cross-linkers were anchored at Cys10 of actin. A second contact site was identified by cross-linking of Cys17 and Cys28 in thymosin beta 4 with the ATP gamma S derivative bound to actin, indicating that the hexamotif of thymosin beta 4 (positions 17-22) is in close proximity (< or = 9.2 A) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta 4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.

Actins↗
PubMed1996

Characterization of natural peptides from bovine tissue using capillary electrophoresis, high performance liquid chromatography, matrix-assisted laser desorption ionization, and Edman degradation.

A combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) spiking, Edman degradation, amino acid analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was applied for the analysis of the primary structure of beta-thymosins. CE has been used to resolve peaks which coelute on HPLC, as well as to help establish the identity of tryptic fragments in the peptide mapping experiments of the two highly homologous beta-thymosins.

Amino Acid Sequence↗