Biomedical subjects

C Weise

Publications and source records attributed to C Weise.

At least 37 records · Page 2Linked to original sources

PubMed1998

Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella.

In this paper, we report the detection, purification and characterization of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI was purified from the hemolymph of last-instar larvae of Galleria mellonella by precipitation with trichloroacetic acid and heat followed by affinity chromatography on a thermolysin-Sepharose column and gel filtration or reverse-phase high-performance liquid chromatography. For the detection of inhibitor activity, a new azocoll assay was established. IMPI was only detectable in larvae that had been injected with bacterial or fungal provocators, suggesting that it is induced nonspecifically during the humoral immune response. Injection of larvae with IMPI rendered them resistant to thermolysin, in quantities that normally would be lethal for them. IMPI was shown to be specific for metalloproteases. The molecular mass of IMPI was determined by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous, owing to different degrees of glycosylation with hexose/hexosamine and deoxyhexose residues. Ten cysteine residues were found in the molecule, and these are presumed to form five disulfide bridges. The amino terminus was blocked, but a partial amino-acid sequence starting from the thermolysin cleavage site was determined; this sequence exhibited no similarity with other known proteins, suggesting that the IMPI represents a new type of protease inhibitor.

Amino Acid Sequence↗

PubMed1998

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of N-acetylglucosamine kinase from rat liver--comparison with UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase.

N-Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase (EC 2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39-kDa subunits. About 25% of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (EC 5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver. The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N-ethylmaleimide, iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol-modifying chemical reagents, sodium meta-periodate, sodium meta-arsenite/2,3-dimercaptopropanol and diazenedicarboxylic acid bis-N,N'-dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDP-GlcNAc 2-epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N-[3H]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.

Acetylglucosamine↗

PubMed1998

Reverse-phase chromatography isolation and MALDI mass spectrometry of the acetylcholine receptor subunits.

A procedure for purifying the Torpedo californica nicotinic acetylcholine receptor subunits is proposed which involves preparative SDS-PAGE followed by reverse-phase HPLC on a C4 column in an acetonitrile-isopropanol system. By this method, the alpha-subunit can be completely separated from the 43-kDa protein which migrates very close to it on SDS-PAGE, and the delta-subunit can be isolated free from the beta-subunit of Na+, K(+)-ATPase comigrating with it on SDS-PAGE. The purity of all acetylcholine receptor subunits thus obtained was verified by Edman degradation and MALDI mass-spectrometric analysis which could be performed quite easily on the HPLC-purified samples. In general, we observed a good correlation between the experimentally determined molecular masses and those calculated from the amino acid sequences and when known, posttranslational modifications (glycosylation and phosphorylation) of individual receptor subunits. Transfer of the isolated receptor subunits into 1% octyl-beta-D-glucopyranoside generates samples suitable for functional studies and enzymatic proteolysis or deglycosylation.

Amidohydrolases↗

PubMed1998

Primary structure of apolipophorin-III from the greater wax moth, Galleria mellonella.

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth, Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.

Amino Acid Sequence↗

PubMed1998

Sertraline in the treatment of panic disorder. A multi-site, double-blind, placebo-controlled, fixed-dose investigation.

BACKGROUND: This study compared the efficacy and safety of sertraline to placebo in treating panic disorder. METHOD: 178 out-patients with panic disorder who exhibited at least four panic attacks during the four weeks prior to screening and three during the two weeks of lead-in were randomly assigned to 12 weeks of double-blind treatment with sertraline (50, 100 or 200 mg) or placebo. RESULTS: Sertraline was superior to placebo in reducing the number of panic attacks, situational attacks, unexpected attacks, limited symptom attacks, and time spent worrying (all P < 0.01) and the Hamilton Anxiety Scale (P < 0.05), although Clinical Global Impression (Improvement) did not significantly differentiate groups at 12 weeks and at end-point. No serious adverse events were associated with sertraline. No dose relationship was found for adverse events; overall drop-out rates were not different for sertraline or placebo, although more sertraline-treated subjects discontinued for adverse events, typically early in the study. Only dry mouth and ejaculation failure (primarily ejaculation delay) were associated significantly with sertraline. CONCLUSIONS: Sertraline was effective and safe in reducing panic attacks. Higher doses were no more effective than the 50 mg dose.

Adolescent↗

PubMed1997

A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Molecular cloning and functional expression of UDP-N-acetyl-glucosamine 2-epimerase/N-acetylmannosamine kinase.

N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stäsche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313-24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both epimerase and kinase activity. Distribution of UDP-GlcNAc 2-epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.

Amino Acid Sequence↗

PubMed1997

Specific binding of HIV-1 envelope protein gp120 to the structural membrane proteins ezrin and moesin.

The observation that HIV in vitro can infect CD4- and Gal-C-negative brain cell lines has stimulated this study to identify alternative gp120-binding proteins on brain cells. HIV-1 gp120 binding proteins of the CD4-negative and Gal-C-negative, non-productively infectable human glioblastoma cell line D54 were purified by affinity chromatography over a gp120-conjugated sepharose column and identified by peptide microsequencing. The binding capacity and specificity of this column was controlled using extracts of CD4-positive cells. Two of seven prominent proteins eluted from the gp120 affinity column specifically bound gp120 in Western blot overlay experiments and were identified by subsequent immunoblotting and microsequencing as ezrin and moesin, members of the ERM (ezrin, radixin, moesin) family of cellular structural membrane proteins. Antibodies to ezrin and moesin specifically recognized the eluted gp120 binding proteins confirming their identification. Ezrin and moesin are structural proteins binding to the cellular membrane and to several cytoskeletal and transmembrane proteins. Our results suggest that ezrin and moesin might play a role as gp160/gp120 binding proteins during the uptake, the assembly or the budding of HIV.

Chromatography, Affinity↗

PubMed1997

Mapping of exposed surfaces of the nicotinic acetylcholine receptor by identification of iodinated tyrosine residues.

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was 125I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the delta-subunit, we identified five iodinated peptides containing the tyrosine residues deltaTyr17, deltaTyr74, deltaTyr365, deltaTyr372, and deltaTyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues deltaTyr17 and deltaTyr74 is extracellular and deltaTyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.

Amino Acid Sequence↗

PubMed1996

The neuronal EF-hand Ca(2+)-binding protein VILIP: interaction with cell membrane and actin-based cytoskeleton.

VILIP is a member of the visinin/recoverin family of neuronal EF-hand Ca(2+)-binding proteins. Cell fractionation revealed cytoplasmic, membrane- and cytoskeleton-associated pools of VILIP. The association with the cytoskeletal protein fraction is Ca(2+)-dependent and may be mediated by direct interaction with actin. This is concluded from the observations that (i) Ca(2+)-loaded recombinant VILIP binds actin in an overlay assay; (ii) in the presence of Ca(2+), beta-actin co-immunoprecipitates with native VILIP from brain extracts, and (iii) actin and VILIP are co-localized in PC12 cells stably transfected with VILIP cDNA. The interaction of VILIP with the cortical cytoskeleton through actin may facilitate a Ca(2+)-dependent recruitment of VILIP to the cell membrane.

Actins↗

PubMed1996

A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores.

In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p-ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis.

Amino Acid Sequence↗

PubMed1995

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

Animals↗

PubMed1995

A new class of photoactivatable and cleavable derivatives of neurotoxin II from Naja naja oxiana. Synthesis, characterisation, and application for affinity labelling of the nicotinic acetylcholine receptor from Torpedo californica.

A new series of photoactivatable and cleavable derivatives of neurotoxin II from the cobra Naja naja oxiana is investigated which can be used for mapping the surface topology of the nicotinic acetylcholine receptor from Torpedo electric tissue. The preparation and characterisation of five toxin derivatives, each with a radioactive 125I-azidosalicylamidoethyl-1,3'-dithiopropyl group in a defined position within the primary structure, are described. The photoinduced cross-linking reaction of the toxin derivatives with membrane-bound receptor is investigated. The photoactivatable group located at position K25 reacts almost exclusively with the delta subunit of the receptor, whereas the K15 derivative reacts with the alpha and beta subunits. The other derivatives did not react with the receptor to any significant extent. It is shown that, with respect to the receptor subunits, the cross-linking pattern depends on the length and chemical nature of the cross-linking group.

Affinity Labels↗

PubMed1995

A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation.

TRAP is a tumor necrosis factor (TNF)-related, 33-kDa type II transmembrane protein almost exclusively expressed on the surface of activated CD4+ T lymphocytes. Interaction of TRAP with CD40 on B cells is of paramount importance for immunoglobulin class switching and subsequent synthesis of IgG, IgA or IgE in vivo. We now provide evidence that activated T cells not only express cell membrane-associated TRAP but also a soluble form of TRAP (sTRAP). After generating monoclonal antibodies against TRAP and establishing a TRAP-specific enzyme-linked immunosorbent assay we were able to detect substantial amounts of sTRAP in the supernatants of activated T cells. The onset and rate of sTRAP release was found to parallel the expression of TRAP on the cell surface. sTRAP, an 18-kDa protein, is generated by proteolytic processing of full-length TRAP in an intracellular compartment. Starting with methionine 113 of full-length TRAP, sTRAP lacks the transmembrane region and a part of the extracellular domain but contains the entire TNF-alpha homology region and can, therefore, bind to CD40. Like other members of the TNF superfamily (e.g. TNF-alpha, Fas/APO-1 ligand), TRAP thus has the potential to be biologically active not only in a transmembrane form but also as a soluble molecule.

Amino Acid Sequence↗

PubMed1995

Chamber-specific expression of human myocardial proteins detected by two-dimensional gel electrophoresis.

High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by computer-assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end-stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2-D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP 0117:M(r)/pI:20.7/ < 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4; SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2-D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.

Amino Acid Sequence↗

PubMed1995

Citric acid extracts a specific set of proteins from isolated cell nuclei.

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA.

Amino Acid Sequence↗

PubMed1995

The prophenoloxidase from the wax moth Galleria mellonella: purification and characterization of the proenzyme.

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. The molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel. In the presence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions. The PPO was shown to be a glycoprotein and its isoelectric point was 6.2. The amino-acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sexta. The activation of the PPO with chymotrypsin was investigated and two proteins of 67 and 50 kDa were found to be products of the proteolytic cleavage. The N-terminus of the G. mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited significant homology with highly conserved sequences found in arthopod hemocyanins and insect storage proteins, which indicates that the PPO belongs to this family.

Amino Acid Sequence↗

PubMed1995

A novel nuclear pore protein Nup82p which specifically binds to a fraction of Nsp1p.

Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsp1p, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A)+RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsp1p participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.

Amino Acid Sequence↗