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W Voelter

Publications and source records attributed to W Voelter.

At least 19 recordsLinked to original sources
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↗
PubMed1995

Capillary electrophoresis, a rapid and sensitive method for routine analysis of apolipoprotein A-I in clinical samples.

A method for the determination of apolipoprotein A-I, the major protein compartment of HDL, in human serum is described. Rapid and easy serum sample preparation, well separated Apo A-I peaks in the human serum electropherogram and good linearity of the peak area vs. concentration plot, covering the range of the clinically relevant Apo A-I serum contents, suggest the introduction of this method routinely in clinical laboratories.

Apolipoprotein A-I↗
PubMed1995

Capillary electrophoresis of human serum proteins and apolipoproteins.

Capillary electrophoresis (CE) analysis of human serum proteins and apolipoproteins is described and compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cellulose acetate membrane electrophoresis. Under optimized CE conditions apolipoproteins I could be determined directly in the serum and quantitated with a linear correlation between peak area and concentration up to a concentration of 100 mg/dL.

Apolipoprotein A-I↗
PubMed1995

The interaction of (1-4)-fragment of thymosin beta 4 with calmodulin-sensitive cAMP phosphodiesterase from hypothalamus.

Evidence was accumulated indicating that cyclic nucleotides are involved in regulation of growth, differentiation and function of lymphoid cells. It was previously shown that the N-fragment (1-4) of thymosin beta 4 (Ac-Ser-Asp-Lys-Pro-OH) inhibits in vivo the entry of cell populations into S-phase. In the course of the study of the interrelationship between the immune and neuroendocrine systems we have found that the tetrapeptide caused incomplete competitive inhibition of hypothalamic calmodulin (CaM)-dependent phosphodiesterase (PDE) stimulated by CaM. In the presence of the peptide, the 20-fold increase of the constant for PDE activation by CaM was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis. The value of the inhibition constant (Ki) amounted to 600 nM. In the absence of CaM, the peptide at saturating concentrations reduced the basal activity of PDE nearly 2- to 3-fold. The effect of the peptide on PDE was noncompetitive with respect to cAMP. The results support our suggestion that the tetrapeptide realizes its effects in the immuno-neuroendocrine system by the mechanism of cyclic nucleotide metabolism.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins.

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-O-methylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M(r) values of 250 and 450 kDa attributed to the two subunits, values of 2.18 x 10(5) daltons and 4.30 x 10(5) daltons were calculated for the polypeptide part of the "light" and "heavy" subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man > or = 3MeGal > GlcNAc > or = GalNAc and Fuc > or = Xyl.

Animals↗
PubMed1995

Functional unit of the Rapana thomasiana (Grosse) (marine snail, gastropod) hemocyanin.

The amino terminal functional unit (domain a) of the Rapana hemocyanin "heavy" structural subunit, designated as Rta, was obtained after limited trypsinolysis of the whole polypeptide chain. Mass spectrometric analysis showed a molecular mass of 49,698 daltons for the electrophoretically homogeneous fragment. Twenty-five amino acid residues were sequenced directly from the N-terminus of Rta, which allowed the location of the domain in the polypeptide chain of the subunit. Physicochemical parameters were determined by absorption and fluorescence spectroscopy and circular dichroism. Comparison with the respective parameters of the whole Rapana hemocyanin showed that the polypeptide backbone folding, binuclear active site and capability of oxygen binding of the isolated functional unit are identical to those of the native hemocyanin. Comparison of N-terminal sequences of functional units from different molluskan hemocyanins and located at different positions revealed some evolutionary relationships.

Amino Acid Sequence↗
PubMed1995

Rational approach to fractionation, isolation, and characterization of polysaccharides from the lichen Cetraria islandica.

Polysaccharides, isolated from the lichen Cetraria islandica, have antimicrobial effectiveness. For pharmaceutical applications the two glucan components lichenan and isolichenan as well as the galactomannan component are of actual interest. Especially the a-glucan isolichenan ist used as an active ingredient in cough lozenges. The conditions for the extraction of the raw material, mainly pH and temperature, have a strong influence on the yield of lichenan, isolichenan, and galactomannan, and also on the amount of tannins in the extract. Target products and also by-products give higher extraction yields with increasing extraction temperatures. Hot water extraction with subsequent fractionation of the extracted polysaccharides by multiple freezing/thawing steps and water removal applying ethanol and ether permitted the isolation of the target polysaccharides in preparative quantities. Tannins were removed by reversed phase chromatography. IR and NMR spectroscopy were used for structural characterization of lichenan and isolichenan. After optimization of the hot water extraction process no significant lower extraction and fractionation yields have been obtained compared to the established tricky DMSO extraction procedure.

Chromatography, High Pressure Liquid↗
PubMed1994

Theoretical and experimental epitope mapping of thymosin beta 4.

Two rabbit polyclonal antisera, one directed against thymosin beta 4 and the other one against the peptide fragment thymosin beta 4 (1-14) were characterised by epitope mapping. Hexapeptides representing the whole sequence of the native peptide and overlapping by one amino acid were synthesised on polystyrene pins. The antigenic determinants were identified in microtitre plates with an ELISA procedure. The polyclonal serum against thymosin beta 4 detected three epitopes (N-terminal, mid-region and C-terminal) whereas the polyclonal serum against the fragment contained only antibodies specific for the N-terminal epitope. These experimental results are consistent with theoretical predictions.

Amino Acid Sequence↗
PubMed1994

Purification, bioactivity, and secondary structure analysis of mouse and human macrophage migration inhibitory factor (MIF).

The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.

Amino Acid Sequence↗
PubMed1994

The sulfoxide of thymosin beta 4 almost lacks the polymerization-inhibiting capacity for actin.

Thymosin beta 4 (T beta 4), a peptide of 43 amino acids, binds to actin monomers and inhibits filament formation. In preparations of T beta 4 from bovine lung tissue, the peptide is accompanied by a derivative in which the methionine residue in position 6 is replaced by its sulfoxide. T beta 4 sulfoxide inhibits actin polymerization to an extent approximately 20-times less than T beta 4. While an equimolar amount of T beta 4 prevented actin polymerization almost completely, polymerization with the corresponding amount of the sulfoxide proceeded in a manner similar to that of pure actin, except for a slight retardation. We showed that the decrease in the inhibitory activity is reflected by a 20-times lower affinity to actin. Interestingly, under non-polymerizing conditions, the affinity of T beta 4 sulfoxide for actin is as high as that of T beta 4 (approximately 1 microM). In accordance with this, no differences were found between T beta 4 and the sulfoxide in cross-linking experiments with the monomer, where both forms of the peptide yielded similar amounts of a 47-kDa band representing conjugates of actin and beta-thymosin, as proved by Western-blotting analysis. Likewise, both, T beta 4 and the sulfoxide retarded the exchange of G-actin-bound nucleotide to similar extents. Although the sulfoxide is presumably a product of autoxidation, it is attractive to speculate that oxidation of the methionine residue in T beta 4 may represent a regulatory switch for starting filament formation in non-muscle cells.

Actins↗
PubMed1994

Polymerization of actin from the thymosin beta 4 complex initiated by the addition of actin nuclei, nuclei stabilizing agents or myosin S1.

Thymosin beta 4 forms a 1:1 complex with actin and thereby prevents polymerization. Rapid formation of filaments from this complex was observed, however, when actin trimers were added. Polymerization can likewise be initiated by the addition of one equivalent of phalloidin or, less effectively, cytochalasin B. Since both toxins, which reportedly support nucleation, have similar effects as the covalently linked actin trimers, it appears that the formation of filaments from the actin-thymosin beta 4 complex depends on the availability of stable actin nuclei. Remarkably, rapid polymerization was also observed if small amounts of myosin S1 were added, suggesting that also myosin, a protein functionally connected with polymeric actin, can serve as a nucleation center. Considering the existence of thymosin beta 4 and related peptides in numerous mammalian tissues, our data suggest that spontaneous formation of microfilaments in non-muscle cells may be regulated at the level of nucleation. Uncontrolled polymerization induced by the formation of phalloidin-stabilized nuclei may explain the acute toxic effects of phalloidin in hepatocytes.

Actins↗
PubMed1994

Radioimmunoassays for the C-terminus of prothymosin alpha and the N-terminus of parathymosin alpha for the measurement of the levels of alpha-thymosins in human cancer.

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107. A radioimmunoassay specific for the N-terminus of human parathymosin alpha, also measuring intact parathymosin alpha in the range of 1-20 pmol and not cross-reacting with prothymosin alpha, was developed using the synthetic peptide [Cys-Aca degrees]-human parathymosin alpha (1-30)-OH as antigen coupled to KLH and the analogue [Tyr-Aca degrees]-human parathymosin alpha (1-10)-OH labelled with 125I as tracer. A major epitope was located in the segment 1-10. These radioimmunoassays, together with a previously established radioimmunoassay for the N-terminus of prothymosin alpha, permitted the identification of the molecular forms of the cross-reactive materials in both normal and neoplastic breast tissue extracts as intact prothymosin alpha and parathymosin alpha. It was also possible to reveal significantly higher levels of both alpha-thymosins in breast cancer tissue compared to the nearby healthy tissue--the mean of 14 samples was over 14-fold higher--suggesting a role of both prothymosin alpha and parathymosin alpha in cell proliferation. The reported radioimmunoassays are expected to facilitate the search for prognostic and/or diagnostic applications of these polypeptides in human cancer.

Aged↗
PubMed1993

Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline.

The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme.

Amino Acid Sequence↗
PubMed1993

Conformation of thymosin beta 4 in water determined by NMR spectroscopy.

The conformational preferences of a 43-amino-acid G-actin-binding peptide, thymosin beta 4, in water at 1, 4 and 14 degrees C, and at pH 3.0 and 6.5 were studied by NMR. NMR showed that thymosin beta 4 lacks a uniquely folded conformation in water. However, some preferential alpha-helical conformations of thymosin beta 4 can be observed in aqueous solutions. The segment at residues 5-16 showed characteristic interactions for conformations in both the beta-strand and alpha-helical regions of the phi-psi space, based on strong C alpha H(i)-NH(i+1) interactions and NH-NH, C alpha H(i)-NH(i+3), and C alpha H(i)-C beta H(i+3) interactions, respectively. At 1-4 degrees C, another segment at residues 31-37 also shows both beta and alpha conformations, forming however a less well-defined helix than the segment at residues 5-16. At 14 degrees C, the conformational population of the helix at positions 5-16 is shifted more towards the random and turn-like structures, whereas the segment at positions 31-37 becomes exclusively a random coil.

Amino Acid Sequence↗
PubMed1993

MIF is a pituitary-derived cytokine that potentiates lethal endotoxaemia.

Cytokines are critical in the often fatal cascade of events that cause septic shock. One regulatory system that is likely to be important in controlling inflammatory responses is the neuroendocrine axis. The pituitary, for example, is ideally situated to integrate central and peripheral stimuli, and initiates the increase in systemic glucocorticoids that accompanies host stress responses. To assess further the contribution of the pituitary to systemic inflammatory processes, we examined the secretory profile of cultured pituitary cells and whole pituitaries in vivo after stimulation with bacterial lipopolysaccharide (LPS). Here we identify macrophage migration inhibitory factor (MIF) as a major secreted protein release by anterior pituitary cells in response to LPS stimulation. Serum analysis of control, hypophysectomized and T-cell-deficient (nude) mice suggests that pituitary-derived MIF contributes to circulating MIF present in the post-acute phase of endotoxaemia. Recombinant murine MIF greatly enhances lethality when co-injected with LPS and anti-MIF antibody confers full protection against lethal endotoxaemia. We conclude that MIF plays a central role in the toxic response to endotoxaemia and possibly septic shock.

Acute-Phase Reaction↗
PubMed1993

Expression of alpha-thymosins in human tissues in normal and abnormal growth.

Radioimmunoassays specific for the N and C termini of human prothymosin alpha and the N terminus of human parathymosin alpha were employed for the measurement of the levels of alpha-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two alpha-thymosins was observed in thymus (prothymosin alpha-rich) and liver (parathymosin alpha-rich). A decline in the levels of both alpha-thymosins was found with age, with prothymosin alpha in thymus showing the sharpest change (15- to 30-fold). The levels of both alpha-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin alpha and parathymosin alpha was 17.9- and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin alpha and parathymosin alpha. These results suggest an in vivo relationship of the expression of alpha-thymosins with the human tissue cell proliferation activity.

Adolescent↗
PubMed1993

Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9.

By reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosin beta 4 (T beta 4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the KD value of the thymosin beta 4 complex as 0.8 microM, a value that is in agreement with previous determinations. In addition to the main compound thymosin beta 4, bovine tissue contains a related peptide, thymosin beta 9 (T beta 9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that T beta 9, similar to T beta 4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a KD value of 1.1 microM the affinity of T beta 9 is in the same range as that of T beta 4, suggesting that T beta 9, like T beta 4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of T beta 9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and T beta 9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)-actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of T beta 9 must be located close to the carboxy-terminus.

Actins↗