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F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 217 records · Page 12Linked to original sources
PubMed1993

Purification and chemical characterization of beta-trace protein from human cerebrospinal fluid: its identification as prostaglandin D synthase.

beta-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23-29 kDa was determined for the polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of beta-trace protein as prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (Thr instead of Ser) was detected at amino acid position 154 of the beta-trace polypeptide chain in the corresponding tryptic peptide. The two N-glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29 and Asn56 bear exclusively complex-type oligosaccharide structures (partially sialylated with alpha 2-3- and/or alpha 2-6-linked N-acetylneuraminic acid) that are almost quantitatively alpha 1-6 fucosylated at the proximal N-acetylglucosamine; approximately 70% of these molecules contain a bisecting N-acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli O18:K1 meningitis isolate.

S fimbrial adhesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newborn meningitis. We recently described the cloning and molecular characterization of a determinant, termed sfaI, from the chromosome of an E. coli urinary tract infection strain. Here we present data concerning a S fimbria-specific gene cluster, designated sfaII, of an E. coli newborn meningitis strain. Like the SfaI complex, SfaII consists of the major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding the subunit proteins of SfaII were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with those of the SfaI complex subunits. Although the sequences of the two major SfaA subunits differed markedly, the sequences of the minor subunits showed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of the meninigitis isolate. These data were confirmed by the isolation and characterization of the SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of SfaI and SfaII revealed that differences between the two Sfa complexes with respect to their capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other than SfaS.

Adhesins, Escherichia coli↗
PubMed1993

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

Adhesins, Escherichia coli↗
PubMed1993

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.

Amino Acid Sequence↗
PubMed1993

ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, carries an ER retention motif.

Overlapping cDNAs encoding the entire human ERGIC-53, a 53 kDa membrane protein of the ER-Golgi intermediate compartment, have been isolated and their nucleotide sequence determined. The isolated cDNA is about 2.7 kb in length. The deduced polypeptide chain for ERGIC-53 consists of 510 amino acids (M(r) 54217) including an N-terminal signal sequence of 30 amino acids, a single putative transmembrane segment of 18 amino acids, and a short cytoplasmic domain of 12 amino acids. Surprisingly, the cytoplasmic segment contains two lysines positioned three and four residues from the C-terminus. Such a double lysine motif is known to function as a retention signal for a group of membrane proteins associated with the ER. Expression of a full-length cDNA of ERGIC-53 in Vero cells revealed intracellular localization similar but not always identical to the endogenously expressed ERGIC-53. The presence of an ER retention motif in a protein of the ER-Golgi intermediate compartment has important implications for the retention mechanism mediated by this signal.

Amino Acid Sequence↗
PubMed1992

Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.

Amino Acid Sequence↗
PubMed1992

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.

Acetylglucosamine↗
PubMed1992

Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli.

The two genes encoding the constituent subunits of the Thermoplasma acidophilum proteasome were expressed in Escherichia coli yielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.

Amino Acid Sequence↗
PubMed1992

Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems.

The tiny injection volumes that are usually necessary to maintain the high efficiency of capillary zone electrophoresis present a major problem if only limited sample amounts are available. To increase the sample load, discontinuous buffer systems were developed that allow the on-column concentration of dilute samples. Injection volumes can be increased in this way by at least a factor of 30. These stacking systems were applied to the analysis of tryptic peptides, to the purity checking of high-performance liquid chromatographic fractions and for the micropreparative separation of peptides with subsequent amino acid sequence analysis.

Amino Acid Sequence↗
PubMed1992

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Amino Acid Sequence↗
PubMed1992

cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily.

The gene encoding human tear prealbumin, a major component of the protein fraction of tear fluid, was cloned from total cDNA of lacrimal gland by polymerase chain reaction using synthetic oligonucleotides derived from N-terminal amino acid sequences of the purified protein. Sequence analysis and a computer-assisted homology search revealed this protein to be a member of the lipocalin superfamily, consisting of hydrophobic-ligand carriers. The deduced amino acid sequence of tear prealbumin shares 58% identity with von Ebner's gland protein from rat, which is supposed to be involved in taste reception. In addition, significant homology has also been found with other members of the lipocalins, e.g. with beta-lactoglobulin. The predicted secondary structure of tear prealbumin resembles that of beta-lactoglobulin in the number and positions of nine beta-sheets and one alpha-helix at the C-terminal part of the protein, thus indicating a three-dimensional structure similar to beta-lactoglobulin. Protein sequencing revealed that the observed electrophoretic heterogeneity of tear prealbumin is due to subtle differences at the N terminus of the protein. The function of tear prealbumin as a lipophilic carrier was further supported by the fact that it binds [3H]retinol in vitro. Although this protein was originally described to be tear-specific, a tear prealbumin-specific antiserum also reacted with proteins of human saliva, sweat, and nasal mucus.

Amino Acid Sequence↗
PubMed1992

Ependymins from the cerebrospinal fluid of salmonid fish: gene structure and molecular characterization.

So far, ependymins (Epds) have been sequenced only from cypriniform fish, and in the past all attempts have failed to characterize, on a molecular level, homologous Epd proteins in higher vertebrates. Therefore, rainbow trout (Oncorhynchus mykiss) Epds, which represent the predominant proteins of the cerebrospinal fluid, have been N-terminally sequenced and the encoding cDNA subsequently cloned using the polymerase chain reaction. Surprisingly, only 40-42% of the amino acids are identical with the corresponding sequences from goldfish (Carassius auratus), and no convincing immunological cross-reactivity is observed with an antiserum raised against purified Epds from C. auratus. O. mykiss possesses two highly homologous genes encoding Epds (Om-I, Om-II), a feature typical of a quasi-tetraploid species. Western analysis, using two specific antibodies against Epds from O. mykiss, revealed a variety of different glycosylation variants. In contrast to C. auratus, Epds from O. mykiss probably do not form disulfide-linked dimers. The structure of one Epd gene and its flanking regions have been determined for the Atlantic salmon (Salmo salar). Six exons were deduced by comparison with the corresponding cDNA sequence from O. mykiss (almost 98% homology with Om-II).

Amino Acid Sequence↗
PubMed1992

Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase.

Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.

Adenosine Diphosphate Ribose↗
PubMed1992

Brefeldin A binds to glutathione S-transferase and is secreted as glutathione and cysteine conjugates by Chinese hamster ovary cells.

Radioactively labeled brefeldin A was used to probe for proteins that interact with this metabolite. The most prominent protein labeled after in vivo incubation of Chinese hamster ovary cells with [3H]brefeldin A turned out to have an apparent molecular mass of 26 kDa. Radioactive peptides derived from the [3H]brefeldin A-labeled protein showed sequence identity with glutathione S-transferases, and immunoblotting after two-dimensional gel electrophoresis confirmed this result. In addition, Chinese hamster ovary cells convert the antibiotic to its glutathionyl and cysteinyl derivatives and secrete them rapidly into the medium. From these findings we conclude that detoxification of the antibiotic in mammalian cells occurs via the glutathion S-transferase system. This may explain the often observed reversibility of brefeldin A's action on the steady state of organelles in mammalian cells.

Amino Acid Sequence↗
PubMed1992

Improved purification, crystallization and primary structure of pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

An improved purification procedure, including nickel chelate affinity chromatography, is reported which resulted in a crystallizable pyruvate:ferredoxin oxidoreductase preparation from Halobacterium halobium. Crystals of the enzyme were obtained using potassium citrate as the precipitant. The genes coding for pyruvate:ferredoxin oxidoreductase were cloned and their nucleotide sequences determined. The genes of both subunits were adjacent to one another on the halobacterial genome. The derived amino acid sequences were confirmed by partial primary structure analysis of the purified protein. The structural motif of thiamin-diphosphate-binding enzymes was unequivocally located in the deduced amino acid sequence of the small subunit.

Amino Acid Sequence↗
PubMed1992

The vegetalizing factor. A member of the evolutionarily highly conserved activin family.

The mesoderm and endoderm inducing vegetalizing factor was partially sequenced after BrCN cleavage. A sequence which is highly conserved in activin A near the C-terminal end was identified. This shows that the factor belongs to the activin family. The activins are not confined to embryos and gonads, but widely distributed in other tissues like calf kidney and calf liver. Functional aspects are discussed.

Activins↗
PubMed1992

Production and characterization of recombinant mouse neurotrophin-3.

Neurotrophin-3 (NT-3) is a neurotrophic-factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 micrograms purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.

Amino Acids↗