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Biomedical subjects

F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 181 records · Page 10Linked to original sources
PubMed1994

The amino acid sequence previously attributed to a protein kinase or a TCP1-related molecular chaperone and co-purified with phytochrome is a beta-glucosidase.

A 60 kDa protein (P60) co-purified with phytochrome was identified as avenacosidase, a beta-glucosidase which is part of the defense system of Avena sativa. An antiserum raised against P60 was used to isolate a cDNA clone coding for the complete amino acid sequence of P60. The cDNA-derived amino acid sequence contained the partial sequences described before for a protein kinase [(1989) Planta 178, 199-206] and for a TCP1-related molecular chaperone [(1993) Nature 363, 644-647] co-purified with phytochrome. We conclude that these activities were related to minor contaminants and that only sequences of avenacosidase had been obtained.

Amino Acid Sequence↗
PubMed1994

DNA replication in vitro by recombinant DNA-polymerase-alpha-primase.

DNA-polymerase-alpha--primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA-polymerase-alpha--primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over-expressed the single polypeptides and co-expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co-expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co-expression of all four subunits yielded large quantities of tetrameric DNA-polymerase-alpha--primase. The p180, p58 and p48 polypeptides were also co-expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA-polymerase-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

Amino Acid Sequence↗
PubMed1994

The (2R)-hydroxycarboxylate-viologen-oxidoreductase from Proteus vulgaris is a molybdenum-containing iron-sulphur protein.

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2-oxocarboxylates at the expense of reduced artificial redox mediators to (2R)-hydroxycarboxylates has been purified to a specific activity of up to 1800 mumol.min-1.mg-1 for the reduction of phenylpyruvate. The membrane-bound non-pyridine nucleotide-dependent enzyme appears in the form of various oligomers of the 80-kDa monomer. The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo-labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis. Permanganate oxidation delivers 0.7 mol pterin-6-carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron-sulphur clusters.

Amino Acid Sequence↗
PubMed1994

Murine protein which binds preferentially to oligo-C-rich single-stranded nucleic acids.

Two single-stranded nucleic acid binding proteins mCBP and mCTBP were identified by means of their binding to a potential recombination hotspot in LTRs of mouse retro-transposons. Both are nuclear proteins of 35 and 55 kDa respectively. mCBP binds preferentially to oligo dC, mCTBP to oligo dCdT. mCBP was purified and its cDNA was isolated and sequenced.

Amino Acid Sequence↗
PubMed1994

The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site.

Like many proteins with a single transmembrane domain the IL-6R exists in a membrane-associated and soluble form. The soluble IL-6R is generated by limited proteolysis of the membranous receptor. This process, also called shedding, is drastically enhanced by PMA, an activator of protein kinase C. The soluble receptor protein was purified to homogeneity from supernatants of COS-7 cells transfected with a cDNA coding for the transmembrane IL-6R. The COOH-terminus of the shed receptor protein was analyzed by carboxypeptidase treatment and subsequent amino acid analysis. The established cleavage site Gln357/Asp358 was extensively altered by point mutations and small deletions to define the structural requirements for cleavage. Although point mutations around the cleavage site reduced shedding of the IL-6R up to fivefold, deletions of 5 or 10 amino acids almost completely abolished shedding. Deletion of the cytoplasmic domain of the receptor had no influence on shedding of the protein. It turned out that a potential N-glycosylation site close to the proteolytic cleavage site of the IL-6R is used. However this N-glycosylation does not affect the efficiency of the shedding process. Furthermore, we demonstrate for the first time that the human IL-6R is constitutively phosphorylated and that this phosphorylation can be stimulated by PMA but is not correlated with shedding of the receptor protein. The knowledge of the mechanism by which the soluble IL-6R is generated will help to identify the processing enzyme involved and to analyze its regulation.

Amino Acid Sequence↗
PubMed1994

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

Amino Acid Sequence↗
PubMed1994

The primary structure of two chlorosome proteins from Chloroflexus aurantiacus.

The complete nucleotide sequence of two chlorosome proteins with apparent molecular weights of M(r) 18,000 and M(r) 11,000 from Chloroflexus aurantiacus have been determined. The two polypeptides were 145 and 97 amino acids long and possessed true molecular masses of 15,545 and 10,820 Da, respectively. Protein chemical sequencing was done in parallel to confirm the primary structure deduced from nucleotide sequencing. By Northern blot analysis of RNA isolated from phototrophically grown cells a transcript of 0.95 kb was detected which is the expected length for a mRNA encoding both genes.

Amino Acid Sequence↗
PubMed1994

Improved method for preparation of lipopolysaccharide-binding protein from human serum by electrophoretic and chromatographic separation techniques.

Recent work has established the importance of serum proteins which interact with endotoxin (lipopolysaccharide, LPS) from Gram-negative bacteria. Thus human monocytes are activated after binding LPS complexed with a serum protein. LPS-binding protein (LBP) is a protein present in both normal and acute phase sera which binds LPS with high affinity. We describe the purification of LBP from human acute phase serum. The purification procedures combine preparative isoelectric focusing (IEF) and either preparative polyacrylamide gel electrophoresis (PAGE) or alternatively an anion-exchange chromatographic step using a Mono Q HR 5/5 column. This allows the isolation of biologically active LBP. LBP was characterized by N-terminal sequence analysis and by measuring the biological activity using flow cytometry (fluorescence-activated cell sorter, FACS) and a luminol enhanced chemiluminescence (LECL) assay.

Acute-Phase Proteins↗
PubMed1994

Maturation of the large subunit (HYCE) of Escherichia coli hydrogenase 3 requires nickel incorporation followed by C-terminal processing at Arg537.

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (delta hycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (delta hycH) chromatographed with radioactivity from 83Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (delta hypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.

Amino Acid Sequence↗
PubMed1994

Moesin: a cell membrane protein linked with susceptibility to measles virus infection.

Measles virus is a highly contagious virus causing acute and persistent diseases in man, the receptor of which is still not well characterized. We have isolated a monoclonal antibody (mAb), designated mAb 119, which specifically inhibits measles virus infection of susceptible cell lines in a dose-dependent manner. This antibody precipitates a protein with an apparent molecular mass of 75 kDa from 125I surface-labeled cells and its epitope is present on human peripheral blood mononuclear cells, human cell lines, and the African green monkey cell line Vero. Affinity chromatography of detergent-solubilized cell membrane proteins over a Sepharose column with covalently bound mAb 119 led to the partial purification of the 75-kDa protein. Preincubation of measles virus with this affinity-purified protein inhibited measles virus infection dose dependently. Amino acid microsequencing of this protein revealed its identity with the human membrane-organizing extension spike protein moesin, a protein intra- and extracellularly associated with the plasma membrane of cells. Subsequently, an antibody raised against purified moesin (mAb 38/87) was also found to specifically inhibit measles virus infection of susceptible cells and confirmed our data obtained with mAb 119. Our data suggest that moesin is acting as a receptor for measles virus.

Amino Acid Sequence↗
PubMed1994

Litomosoides carinii microfilarial sheaths: partial amino acid sequences of several major polypeptide constituents.

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of fish neurolin: a growth-associated cell surface protein and member of the immunoglobulin superfamily in the fish retinotectal system with similarities to chick protein DM-GRASP/SC-1/BEN.

We have used the polymerase chain reaction to isolate cDNAs coding for goldfish and zebrafish neurolin, a previously identified 86 kDa cell surface glycoprotein in the goldfish visual system. Sequence analysis demonstrates that neurolin belongs to the immunoglobulin superfamily and is 51% similar to the chick cell adhesion molecule DM-GRASP, SC-1, BEN. Northern analysis with a riboprobe coding for the C-terminus of neurolin detected two mRNAs of 3.7 kb and 3.3 kb in both embryonic and adult goldfish. Several monoclonal and polyclonal antibodies were generated against immunopurified goldfish neurolin and two are shown to crossreact with zebrafish proteins. Both antibodies identify a zebrafish protein of the same molecular weight as goldfish neurolin on immunoblots. Immunohistochemical studies with these antibodies in the zebrafish retinotectal system demonstrate labeling on young ganglion cells and growing retinal axons in a pattern similar to that found in goldfish. The similarity of neurolin to a known cell adhesion molecule, its expression on developing retinal ganglion cells and axons in both embryos and adult fish, and its re-expression during retinal axon regeneration in the goldfish suggests that neurolin is important during axonal growth in the fish central nervous system.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1994

Adenylylsulphate reductase from the sulphate-reducing archaeon Archaeoglobus fulgidus: cloning and characterization of the genes and comparison of the enzyme with other iron-sulphur flavoproteins.

Adenylylsulphate (adenosine-5'-phosphosulphate, APS) reductase from the extremely thermophilic sulphate-reducing archaeon Archaeoglobus fulgidus is an iron-sulphur flavoprotein containing one non-covalently bound flavin group, eight non-haem iron and six labile sulphide atoms per molecule. Reevaluation of the enzyme structure revealed the presence of two different subunits with molecular masses of 80 and 18.5 kDa. The subunits are arranged in an alpha 2 beta subunit structure. We have cloned and sequenced a 2.7 kb segment of DNA containing the genes for the alpha and beta subunits, which we designate aprA and aprB, respectively. The two genes are separated by 17 bp and localized in the order aprBA. While a putative promoter could not be identified in the vicinity of aprBA a probable termination signal was found just downstream of the translation stop codon of aprA. The codon usage for aprBA shows strong preferences for G and C in the third codon position. aprA encodes a 73.3 kDa polypeptide, which shows significant overall similarities with the flavoprotein subunits of the succinate dehydrogenases from Escherichia coli and Bacillus subtilis and the corresponding flavoprotein of E. coli fumarate reductase. Part of the homologous peptide stretches could be assigned to domains that are involved in the binding of the substrate or of the FAD prosthetic group. aprB encodes a 17.1 kDa polypeptide representing an iron-sulphur protein, seven cysteine residues of which are arranged in two clusters typical of ligands of the iron-sulphur centres in ([Fe3S4][Fe4S4]) 7-Fe ferredoxins.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence of the Wolinella succinogenes flagellin, which contains in the antigenic domain two conserved regions also present in Campylobacter spp. and Helicobacter pylori.

Wolinella succinogenes possesses one polar flagellum, which shows a characteristic surface pattern of parallel lines along the axis of the filament in electron microscopic images. We determined the gene sequence of the Wolinella flagellin, which is, as in most other bacteria, the only structural component of the filament. Sequence comparison with other members of the Proteobacteria revealed two highly conserved regions in the central part of the flagellin molecule among Campylobacter spp. and Helicobacter pylori, an area that had previously been described as highly variable. Similar surface patterns are found in related polarly flagellated bacteria, but not in Escherichia coli and Bacillus subtilis, which also lack these conserved regions.

Amino Acid Sequence↗
PubMed1994

The interleukin-6-activated acute-phase response factor is antigenically and functionally related to members of the signal transducer and activator of transcription (STAT) family.

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotropic factor are a family of cytokines and neuronal differentiation factors which bind to composite plasma membrane receptors sharing the signal transducing subunit gp130. We have shown recently that IL-6 and leukemia inhibitory factor rapidly activate a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation, which then binds to IL-6 response elements of various IL-6 target genes. Here we demonstrate that APRF is activated by all cytokines acting through gp130 and is detected in a wide variety of cell types, indicating a central role of this transcription factor in gp130-mediated signaling. APRF activation is also observed in vitro upon addition of IL-6 to cell homogenates. Protein tyrosine kinase inhibitors block both the tyrosine phosphorylation and DNA binding of APRF. The factor was purified to homogeneity from rat liver and shown to consist of a single 87-kDa polypeptide, while two forms (89 and 87 kDa) are isolated from human hepatoma cells. As reported earlier, the binding sequence specificity of APRF is shared by gamma interferon (IFN-gamma) activation factor, which is formed by the Stat91 protein. Partial amino acid sequence obtained from purified rat APRF demonstrated that it is likely to be related to Stat91. In fact, an antiserum raised against the amino-terminal portion of Stat91 cross-reacted with APRF, suggesting the relatedness of APRF and Stat91. Altogether, these data indicate that APRF belongs to a growing family of Stat-related proteins and that IFN-gamma and IL-6 use similar signaling pathways to activate IFN-gamma activation factor and APRF, respectively.

3T3 Cells↗
PubMed1994

Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

Adenosine Triphosphate↗
PubMed1994

The most abundant soluble basic protein of the stylar transmitting tract in potato (Solanum tuberosum L.) is an endochitinase.

An abundant, pistil-specific basic protein has been purified and characterized from potato (Solanum tuberosum L.). A polymerase chain reaction (PCR) probe was generated for the corresponding gene using oligonucleotides based on internal peptide sequences of the protein, and the PCR probe was further employed to isolate cDNA and genomic clones. The sequence of the gene exhibits up to 70% similarity to previously described endochitinase class 1a protein sequences, and the purified protein possesses chitinase (poly[1, 4-(N-acetyl-beta-D-glucosaminide)] glucanohydrolase, EC 3.2.1.14) activity. The protein, termed SK2, has been located by immunocytochemistry to the intercellular matrix of the stylar transmitting tract. Immunoblot analysis has shown SK2 to be distinct from the wound-induced chitinases of potato. The SK2-class of chitinase is restricted in its distribution within the Solanaceae to the sub-family Solanoidae, which includes cultivated tomato and potato species. It was absent from the Cestroidae species tested (Petunia hybrida, Nicotiana tabacum). A role for SK2 endochitinase in protecting the ovary against pollen-tube-mediated pathogen ingress is proposed.

Amino Acid Sequence↗