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F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 307 records · Page 17Linked to original sources
PubMed1989

Molecular characterization of an ependymin precursor from goldfish brain.

Ependymins are thought to be implicated in fundamental processes involved in plasticity of the goldfish CNS. Gas-phase sequencing of purified ependymins beta and gamma revealed that they share the same N-terminal sequence. Each sequence displays microheterogeneities at several positions. Based on the protein sequences obtained, we constructed synthetic oligonucleotides and used them as hybridization probes for screening cDNA libraries of goldfish brain. In this article we describe the full-length sequence of a mRNA encoding a precursor of ependymins. A cleavable signal sequence characteristic of secretory proteins is located at the N-terminal end, followed directly by the ependymin sequence. Also, two potential N-glycosylation sites were detected. A computer search revealed that ependymins form a novel family of unique proteins.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli.

The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).

Adhesins, Escherichia coli↗
PubMed1989

S-layer protein gene of Acetogenium kivui: cloning and expression in Escherichia coli and determination of the nucleotide sequence.

Acetogenium kivui is anaerobically growing thermophilic bacterium with a gram-positive type of cell wall structure. The outer surface is covered with a hexagonally packed surface (S) layer. The gene coding for the S-layer polypeptide was cloned in Escherichia coli on two overlapping fragments by using the plasmid pUC18 as the vector. It was expressed under control of a cloned Acetogenium promoter or the lacZ gene. We determined the complete sequence of the structural gene. The mature polypeptide comprises 736 amino acids and is preceded by a typical procaryotic signal sequence of 26 amino acids. It i weakly acidic, weakly hydrophilic, and contains a relatively high proportion of hydroxyamino acids, including two clusters of serine and threonine residues. An N-terminal region of about 200 residues is homologous to the N-terminal part of the middle wall protein, one of the two S-layer proteins of Bacillus brevis, and there is also an internal homology within the N-terminal region of the A. kivui polypeptide.

Amino Acid Sequence↗
PubMed1989

Substitution of a pentalenolactone-sensitive glyceraldehyde-3-phosphate dehydrogenase by a genetically distinct resistant isoform accompanies pentalenolactone production in Streptomyces arenae.

Pentalenolactone (PL), an antibiotic produced by Streptomyces arenae, is a potent inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The producer strain contains different isoforms of GAPDH: a PL-sensitive enzyme on nonproduction media and a PL-insensitive enzyme on production media. After induction of PL synthesis, the sensitive GAPDH disappears parallel to the disappearance of its activity, as shown by Western (immunoblot) hybridization. The two isoenzymes exhibit little immunological cross-reactivity and differ in size, amino acid composition, and several amino acid residues of their amino termini. Two different types of plasmids from a S. arenae genomic library, named pBRPLR1 and pBRPLR2, were cloned in Escherichia coli by selection for enhanced PL resistance. Both contain a GAPDH structural gene. Plasmid pBRPLR1 increases E. coli PL tolerance 7-fold, and plasmid pBRPLR2 increases it 30-fold. GAPDH from pBRPLR1 transformants shows biphasic PL inactivation kinetics. These cells contain PL-sensitive GAPDH from both E. coli and S. arenae. GAPDH from pBRPLR2 transformants tolerates higher PL concentrations than either E. coli or S. arenae PL-sensitive GAPDH but is less resistant than S. arenae PL-insensitive GAPDH. Nondenaturing polyacrylamide electrophoresis showed this GAPDH to be a hybrid of E. coli and S. arenae PL-insensitive GAPDH. The hybrid enzyme could be purified to homogeneity. Induction of the lacZ promoter of pUC subclones of both GAPDH genes had only a small effect on raising the level of intracellular GAPDH.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of cytolysins from Listeria monocytogenes serovar 4b and Listeria ivanovii.

Several exoproteins from Listeria monocytogenes serovar 4b (NCTC 10527) and Listeria ivanovii (ATCC) 19119, SLCC 2379), respectively, have been purified to homogeneity by thiol-disulfide exchange chromatography and gel filtration. Both strains produce a haemolytic/cytolytic protein of Mr 58 kDa, which has all the properties of a SH-activated cytolysin, the prototype of which is streptolysin O (SLO), and this protein has therefore been termed listeriolysin O (LLO). In addition a protein of Mr 24 kDa from culture supernatants of L. ivanovii co-purified with LLO. The N-terminal aminoacid sequences of both proteins from L. ivanovii have been determined. By mutagenesis with transposons of Gram-positive origin (Tn916 and Tn1545), which have been introduced via conjugation into L. ivanovii, several phenotypic mutants (altered haemolysis on sheep blood agar or lecithinase-negative) were obtained. Results on the properties of these mutants will be presented.

Amino Acid Sequence↗
PubMed1988

Purification methods for the sequence-specific DNA-binding protein nuclear factor I (NFI)--generation of protein sequence information.

The paper describes a potent purification method, preparative gel retention, for the purification of sequence-specific DNA-binding proteins. This procedure exploits the sequence-specific DNA-binding affinity of such proteins for their enrichment, comparable to recognition site DNA affinity chromatography. The method was employed to obtain a pure preparation of nuclear factor I (NFI) from porcine liver from which sequences of partial peptides could be obtained. Oligonucleotide probes derived from these amino-acid sequences were used to identify genomic and cDNA clones of NFI.

Amino Acid Sequence↗
PubMed1988

Purification of yolk protein 2 of Drosophila melanogaster and identification of its site of tyrosine sulfation.

We have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 of Drosophila melanogaster. Yolk proteins were purified from [35S]sulfate-labeled flies, and yolk protein 2 was separated from yolk protein 1 and yolk protein 3 by preparative two-dimensional polyacrylamide gel electrophoresis. After digestion of yolk protein 2 with trypsin and reversed-phase high performance liquid chromatography, the sulfate label was recovered in two distinct sulfopeptides which, however, had identical NH2-terminal sequences and contained 3 tyrosine residues each. After chymotryptic digestion of the two tryptic sulfopeptides, the sulfate label was recovered in one sulfopeptide which contained a single tyrosine residue. NH2-terminal sequencing showed that this tyrosine residue corresponded to tyrosine 172 of the yolk protein 2 precursor (Hung, M.-C., and Wensink, P. C. (1983) J. Mol. Biol. 164, 487-492) in the sequence Glu-Thr-Thr-Asp-Tyr(S)-Ser-Asn-Glu-Glu. This insect tyrosine sulfation site is very similar to the known vertebrate tyrosine sulfation sites in terms of amino acid composition and secondary structure. In the accompanying paper (Friederich, E., Baeuerle, P. A., Garoff, H., Hovemann, B., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14930-14938), we report on the expression of Drosophila yolk protein 2 in mouse fibroblasts and show the in vivo sulfation of tyrosine 172 by the vertebrate tyrosylprotein sulfotransferase.

Amino Acid Sequence↗
PubMed1988

A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins.

A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.

Amino Acid Sequence↗
PubMed1988

Cloning and sequencing of a Clostridium perfringens sialidase gene.

Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA. Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid. A synthetic oligonucleotide representing the N-terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C. perfringens genome. The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced. The structural gene encodes 382 amino acids representing an Mr of 42770. A hydrophobic leader sequence is absent. Upstream from the initiation codon ATG, a GA-rich region is found and considered as the Shine-Dalgarno sequence. Homology with the N-terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.

Amino Acid Sequence↗
PubMed1988

The cDNA clone for strictosidine synthase from Rauvolfia serpentina. DNA sequence determination and expression in Escherichia coli.

The cDNA clone for strictosidine synthase, the enzyme which catalyzes the stereospecific condensation of tryptamine with secologanin to form the key intermediate in indole alkaloid biosynthesis, strictosidine, has been identified with a synthetic oligodeoxynucleotide hybridization probe in a lambda gt11 cDNA library of cultured cells of Rauvolfia serpentina. The DNA has been sequenced, revealing an open reading frame of 1032 base pairs encoding 344 amino acids. The sequence of 60 nucleotides in the 5'-flanking region has been determined by primer extension analysis. The encoded protein has been expressed in E. coli DH5 as detected by immunoblotting of protein extracts with antibodies raised against the native enzyme.

Amino Acid Sequence↗
PubMed1988

Human T cell specific proteinase (HuTSP) is encoded by the T cell and natural killer cell specific human Hanukah factor (HuHF) gene.

HuTSP, a serine proteinase which is specifically associated with activated t lymphocytes, was purified to homogeneity and characterized. N-terminal amino acid sequencing revealed identity with a sequence predicted from the human Hanukah factor (HF) gene, which was isolated from a human T cell cDNA library. The dimeric structure of HuTSP, together with its extensive sequence homologies with the murine T cell specific proteinase, MTSP-1, suggests phylogenetic conservation of this serine proteinase family.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5'-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

Amino Acid Sequence↗
PubMed1988

Identification of [hydroxyproline3]-lysyl-bradykinin released from human kininogens by human urinary kallikrein.

The types of kinins released from purified native, single chain human high and low molecular mass kininogens (HMMKs and LMMKs, respectively) by purified human urinary kallikrein were separated by reverse-phase HPLC and quantitated by the rat uterus bioassay. [Hyp3]-lysyl-bradykinin, a recently discovered kinin, represented up to 58% of the biological activity released from 4 individual HMMK preparations purified from 4 different healthy volunteers. In contrast, the majority of the biological activity released from LMMKs purified from pooled plasma was identified as Lys-bradykinin and [Hyp3]-lysyl-bradykinin represented only 6.4 +/- 3.8%. These findings indicate posttranslation hydroxylation of human kininogens and suggest a preference of HMMKs for this modification.

Amino Acid Sequence↗
PubMed1988

Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains.

cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.

Actins↗
PubMed1988

Induction of T cell serine proteinase 1 (TSP-1)-specific mRNA in mouse T lymphocytes.

An oligonucleotide probe corresponding to nucleotides of a cDNA encoding the T cell-associated proteinase 1 (TSP-1) was chosen to study the induction and expression of TSP-1-specific transcripts in mouse T lymphocytes and tissues. We demonstrate that TSP-1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocytes. Expression of the TSP-1 gene was observed in T lymphocytes in vitro in response to either phorbolester (phorbol 12-myristate 13-acetate), Ca2+ ionophore (A23187), lectin or alloantigen. In general, TSP-1 mRNA appeared and peaked later compared to interleukin 2 transcripts. Furthermore, TSP-1 mRNA was inducible in vitro in both Ly-2+ and L3T4+ lymphocyte populations treated with alloantigen and/or lectin. The transcription of the TSP-1 gene was always accompanied by the expression of proteinase activity. High expression of TSP-1 transcripts was also observed in in vivo derived T effector cells specific for lymphocytic choriomeningitis virus. However, TSP-1 mRNA was predominantly associated with virus-specific Ly-2+ T cells and correlated with their proteinase and cytolytic activities. The data suggest that TSP-1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.

Amino Acid Sequence↗
PubMed1988

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis.

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.

Amino Acid Sequence↗