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F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 91 records · Page 5Linked to original sources
PubMed1997

The cDNA sequences of cytochrome c oxidase subunit VIa from carp and rainbow trout suggest the absence of isoforms in fishes.

The cDNAs of subunit VIa of cytochrome c oxidase from rainbow trout liver and carp heart are presented, revealing 82% identity of their deduced amino acid sequences. The two cDNAs are evolutionary equally distant from the livertype (VIaL) and heart-type (VIaH) of mammalian subunit VIa. The data suggest that in ectotherm fishes no isoforms of subunit VIa occur, and that the postulated tissue-specific mechanism of thermogenesis in mammals, based on interaction of ATP with subunit VIaH (Frank, V. and Kadenbach, B. (1996) FEBS Lett. 382, 121-124), is absent.

Amino Acid Sequence↗
PubMed1997

cDNA cloning of porcine p42IP4, a membrane-associated and cytosolic 42 kDa inositol(1,3,4,5)tetrakisphosphate receptor from pig brain with similarly high affinity for phosphatidylinositol (3,4,5)P3.

We previously identified a 42 kDa Ins(1,3,4,5)P4 (InsP4) receptor protein (p42IP4) in brain membranes from several species. Here the cDNA sequence of p42IP4 was obtained by PCR using degenerate primers derived from peptide sequences of proteolytic fragments of the porcine protein and by subsequent screening of a pig brain cDNA library. The derived peptide sequence of 374 amino acids for porcine p42IP4 is 45 amino acids shorter at the C-terminus than centaurin-alpha from rat (84% homology) and has a calculated molecular mass of 43 kDa. From the InsP4 binding activity present in brain tissue homogenate about 25% is found in the cytosolic fraction and 75% associated with microsomes. Both activities are due to p42IP4 since (i) a peptide-specific antiserum recognizing specifically p42IP4 labels the InsP4 receptor protein in membranes and in the cytosol, (ii) the antiserum immunoprecipitates both the membrane protein and the cytosolic protein of 42 kDa, (iii) the InsP4 binding activity released by high salt or by alkaline extraction from membranes is identified immunologically as the 42 kDa protein, and (iv) the affinity for InsP4 and specificity for various inositolphosphates are similar for the membrane-associated and for the soluble p42IP4. The functional importance of p42IP4 is highlighted by the identical affinity for InsP4 and for phosphatidylinositol (3,4,5)P3 (Ki = 1.6 and 0.9 nM, respectively). Thus, the InsP4 receptor, apparently a peripheral membrane protein, which exists also as a cytosolic protein can transfer the signals mediated by InsP4 or by PtdInsP3 between membranes and cytosolic compartment.

Amino Acid Sequence↗
PubMed1997

The primary structure of the Cl(-)-translocating ATPase, b subunit of Acetabularia acetabulum, which belongs to the F-type ATPase family.

The genes possibly encoding the b subunit (50 kDa) of the Cl(-)-translocating ATPase of Acetabularia acetabulum were cloned from total RNA and from poly(A)+ RNA and sequenced. The deduced amino acid sequence of the open reading frame consisted of 478 amino acids and showed high similarity to the beta subunit of chloroplast F1-ATPase. Gene fragments encoding the putative beta subunit of chloroplast F1- (273 bp) and mitochondrial F1-ATPases (332 bp) were also cloned from A. acetabulum and sequenced, respectively. The deduced amino acid sequence of the chloroplast F1-ATPase showed 92.5% identity to be primary structure of the b subunit of the Cl(-)-translocating ATPase, while the nucleotide sequences were 79.9% identical. The deduced amino acid sequence of the latter was 77.3% identical to that of the b subunit of the Cl(-)-translocating ATPase and the nucleotide sequences were 67.5% identical. By Northern analysis, these three beta-like genes were demonstrated to be transcribed with different sizes of RNA species. A putative chloroplast F1-beta fragment also hybridized with chloroplast DNA isolated from the organism.

Acetabularia↗
PubMed1997

Molecular characterization of E587 antigen: an axonal recognition molecule expressed in the goldfish central nervous system.

The E587 antigen (Ag) is a 200-Kd membrane glycoprotein originally identified by a monoclonal antibody on new and regenerating retinal ganglion cell axons in the adult goldfish. We report the isolation of cDNAs encoding the E587 Ag and identify it as a member of the L1 family of cell adhesion molecules (CAMs). The predicted amino acid sequence of E587 Ag shows an approximately equal identity (40%) to mouse L1, chick neuron-glia CAM, and chick neuron-glia-related CAM. Although the overall similarity is low, there is a high conservation of structural domains and specific sequence motifs. Wholemount in situ hybridizations were performed on goldfish between 34 hours and 3 days postfertilization (pf). A dramatic increase in E587 Ag mRNA was observed between 34 and 48 hours pf. The expression of E587 Ag mRNA in neurons shortly precedes axonogenesis. A marked decrease in expression occurs by 3 days pf, when the axonal scaffold has already been established. Wholemount immunohistochemistry on embryos demonstrates expression of E587 Ag on all major tracts. E587 Ag is absent from mature retinal ganglion cell axons, but its expression is induced by optic nerve transection. A corresponding induction of E587 Ag mRNA in retinal ganglion cells is shown by in situ hybridization. Furthermore, E587 Ag mRNA was detected in the optic nerve, which suggests that nonneuronal cells also express this molecule. E587 Ag was previously shown to promote retinal axon fasciculation and outgrowth in young fish and to mediate axon-glial interactions in vitro. The expression pattern and developmental regulation of E587 Ag in the central nervous system, its reexpression in retinal ganglion cells following optic nerve transection, and its relation to the L1 family indicate that E587 Ag functions as a cell recognition molecule important during axonal growth and regeneration.

Amino Acid Sequence↗
PubMed1997

Subcellular fractionation of polarized epithelial cells and identification of organelle-specific proteins by two-dimensional gel electrophoresis.

Protein targeting and sorting is accomplished by complex vesicular transport processes that are tightly regulated within a cell. This is especially important for epithelial cells because correct delivery of newly synthesized proteins as well as recycling and sorting of internalized membrane proteins is essential for the establishment and preservation of cellular polarity. Many transport events, linking various subcellular compartments, have been analyzed, but many transport mechanisms still remain unresolved. In this study we attempted to identify proteins specifically associated with distinct organelles in murine mammary epithelial cells (EpH4). We isolated subcellular compartments by continuous sucrose gradient centrifugation in order to further analyze their protein composition by high-resolution two-dimensional gel electrophoresis (2-DE). The successful separation of late endosomes (LE), early endosomes (EE) and most of the rough endoplasmic reticulum (RER) was confirmed by subsequent analysis of gradient fractions for compartment-specific enzymes and marker proteins. Both Golgi and plasma membrane (PM) were found to partially co-purify with EE in such gradients. Characteristic polypeptide patterns were revealed on 2-DE gels for fractions enriched in membranes of different origin. Based on improved sample preparation and loading techniques (this issue, C. Pasquali et al., Electrophoresis, 1997, 18, 2573-2581), we were able to identify several proteins by immunoblotting or microsequencing of Coomassie-stained spots. This will be the basis for a further characterization of organelle-specific molecules in epithelial cells as well as for the establishment of a 2-DE reference map of membrane proteins from murine mammary epithelium.

Animals↗
PubMed1997

Autoantibodies in sera of patients with myocarditis: characterization of the corresponding proteins by isoelectric focusing and N-terminal sequence analysis.

In myocarditis an antigen-specific immune response to cardiac epitopes has been demonstrated by several investigators. In 54 patients with histologically proven myocarditis, autoantibodies to cardiac tissue were observed in 73% of patients utilizing the indirect immunofluorescence test with human myocardium and adult heterologous cardiocytes. By immunoblot, 44% of sera from patients reacted with cardiac tissue. These antibodies were directed preferentially against proteins with the molecular weight of 43 up to 67 kDa. Three particular proteins were identified by the use of two-dimensional immunoblot and further characterized by amino acid sequence analysis. To characterize the epitopes recognized by the autoantibodies isoelectric focusing followed by SDS-PAGE was used to separate the complex mixture of proteins from human heart. Immunoblot analysis of antigens revealed proteins ranging from a size of 30-67 kDa at isoelectric points of 6.5-8.5 to be of particular interest. Five of these proteins have now been analyzed by N-terminal amino acid sequencing (Edman degradation). One was found to be creatine kinase and one was identified as a yet unknown protein. Three proteins were N-terminally blocked and were investigated further by enzymatical digestion, followed by separation of the usually complex peptide mixtures on HPLC. One of the peptides was found to be dihydrolipoamide dehydrogenase, a membrane enzyme, and one was identified as a sarcomere specific creatine kinase. Because these proteins are intracullularly located enzymes, their pathogenetic role as antigens for the autoantibodies remains to be elucidated further.

Adult↗
PubMed1997

Properties of prostacyclin synthase.

Prostacyclin synthase (PGIS) was isolated from bovine aortic microsomes after detergent solubilisation following purification by DEAE-Sephacel, immobilized metal affinity, and hydroxy apatite chromatography. The homogenous protein exhibited spectral characteristics of a heme-thiolate protein (P450) like the enzyme purified earlier from porcine microsomes and had an apparent mass of 52 kDa on SDS/PAGE. Three peptides from an endoproteinase Lys-C digest were isolated and sequenced. An antiserum was prepared from rabbits and purified by affinity chromatography. This allowed Western blots of microsomes from cultured endothelial cells. After treatment with IL-1 the activity of the cells in producing 6-keto-PGF1 alpha increased about threefold over 27 h which was accompanied by an increase in PGIS mass. A monoclonal antibody was used to set up an ELISA which served for the quantitation of PGIS in bovine tissues.

Amino Acid Sequence↗
PubMed1997

Fritz: a secreted frizzled-related protein that inhibits Wnt activity.

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

Amino Acid Sequence↗
PubMed1997

High-sensitivity peptide mapping by micro-LC with on-line membrane blotting and subsequent detection by scanning-IR-MALDI mass spectrometry.

A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.

Chromatography, High Pressure Liquid↗
PubMed1997

Genes encoding two different beta-glucosidases of Thermoanaerobacter brockii are clustered in a common operon.

A 5.9-kb fragment of chromosomal DNA coding for beta-glucosidase activity of the thermophilic anaerobe Thermoanaerobacter brockii was sequenced. Two genes, cglT and xglS, encoding a cellodextrin-cleaving beta-glucosidase and a xylodextrin-degrading xylo-beta-glucosidase, respectively, were located directly adjacent to each other. The 5' region contained two additional genes, cglF and cglG, whose products exhibited similarity to integral membrane proteins of metabolite transport systems. The two beta-glucosidases, CglT and XglS, with deduced molecular masses of 52 and 81 kDa, belong to different families of glycosyl hydrolases. Both enzymes were overexpressed in Escherichia coli and could be detected after protein gel electrophoresis and activity staining. The enzyme CglT was purified by fast protein liquid chromatography and identified by N-terminal sequencing. The enzyme was thermostable at 60 degrees C for at least 24 h, and the temperature optimum was 75 degrees C. The ki for glucose inhibition was calculated to 200 mM. The enzyme released glucose from the nonreducing end of beta-1,4-cello oligomers as well as from various disaccharides. CglT was active on glucosides, galactosides and on fucosides, while XglS cleaved beta-glucosides and beta-xylosides as well. The cglT gene was also expressed in Bacillus subtilis, and the enzyme was mainly intracellular during exponential growth but was efficiently released into the supernatant after cultures entered the stationary phase.

Amino Acid Sequence↗
PubMed1997

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.

Amino Acid Sequence↗
PubMed1997

CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus.

3T3 Cells↗
PubMed1997

Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-myc genes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other's binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-myc P2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.

Animals↗
PubMed1997

Reggie-1 and reggie-2, two cell surface proteins expressed by retinal ganglion cells during axon regeneration.

Fish--in contrast to mammals--regenerate retinal ganglion cell axons when the optic nerve is severed. Optic nerve injury leads to reexpression of proteins, which typically are first expressed in newly differentiated retinal ganglion cells and axons. Here we identified two new proteins of fish retinal ganglion cells, reggie-1 and reggie-2, with monoclonal antibody M802 and molecular cloning techniques. In normal fish, M802 stained the few retinal axons derived from newborn ganglion cells which in fish are added lifelong to the retinal margin. After optic nerve injury, however, M802 labeled all retinal ganglion cells and retinal axons throughout their path into tectum. Consistent with M802 staining, reggie-1 and reggie-2 mRNAs were present in lesioned retinal ganglion cells, as demonstrated by in situ hybridization, but were not detectable in their normal mature counterparts. In western blots with membrane proteins of the adult goldfish brain, M802 recognizes a 48x10(3) Mr protein band. At the amino acid level, 48x10(3) Mr reggie-1 and reggie-2 are 44% identical, lack transmembrane and membrane anchor domains, but appear membrane associated by ionic interactions. Reggie-1 and reggie-2 are homologous to 35x10(3) Mr ESA (human epidermal surface antigen) but are here identified as neuronal surface proteins, present on newly differentiated ganglion cells at the retinal margin and which are reexpressed in mature ganglion cells upon injury and during axonal regeneration.

Amino Acid Sequence↗
PubMed1996

Two potential indole-3-acetaldehyde dehydrogenases in the phytopathogenic fungus Ustilago maydis.

The phytopathogenic basidiomycetc Ustilago maydis produces indole-3-acetic acid (IndCH2COOH) and indole-3-pyruvic acid (Ind-Prv) from tryptophan. Indole-3-acetaldehyde (IndCH2CH2O) is the common intermediate in the conversion of Ind-Prv and tryptamine to IndCH2COOH. We purified an enzyme (Iad1) from U. maydis that catalyzes the NAD(+)-dependent conversion of IndCH2CH2O to IndCH2COOH and isolated corresponding cDNA and genomic clones. The identity of the cDNA clone was confirmed by expression in Escherichia coli and demonstration of enzymatic activity. In U. maydis, iad1-null mutants were generated by gene replacement. The ability to convert IndCH2CH2O to IndCH2COOH was at least 100-fold reduced in U. maydis iad1-null mutants grown in medium with glucose as carbon source. However, the iad1-null mutants were not diminished in their capacity to produce IndCH2COOH from tryptophan, indicating that IndCH2COOH formation from tryptophan apparently proceeds in the absence of IndCH2CH2O dehydrogenase activity under these conditions. Iad1 expression was strongly induced during growth on ethanol while under these conditions iad1-null mutants were unable to grow. This reveals that iad1 is primarily engaged in the conversion of ethanol to acetate. In iad1-null mutants we detected an additional NAD(+)-dependent IndCH2CH2O dehydrogenase activity that was induced during growth on L-arabinose but repressed in the presence of D-glucose. In arabinose-containing medium the conversion of tryptophan to IndCH2COOH was approximately 5-fold reduced in wild-type strains but 10-15-fold reduced in iad1-null mutant strains compared to IndCH2COOH formation in glucose-containing medium. In addition, the formation of Ind-Prv from tryptophan was abolished in wild-type and iad1-null mutant strains. During growth on arabinose, the conversion of tryptamine to IndCH2COOH was strongly favored suggesting that the glucose-repressible IndCH2CH2O dehydrogenase is required to convert IndCH2CH2O derived from tryptamine to IndCH2COOH.

Aldehyde Dehydrogenase↗
PubMed1996

Tricorn protease (TRI) interacting factor 1 from Thermoplasma acidophilum is a proline iminopeptidase.

Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H-Pro-AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N-terminus. The pip gene encodes a 293 amino acid residue protein with a molecular mass of 33,487 Da. By means of site-directed mutagenesis we identified Ser105 and His271 as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.

Amino Acid Sequence↗
PubMed1996

Tricorn protease--the core of a modular proteolytic system.

Large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. A tricorn-shaped protease was isolated from the archaeon Thermoplasma and was shown to form a multisubunit proteolytic complex. The 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. Tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller proteins, it displayed multicatalytic activities.

Amino Acid Sequence↗