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F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 109 records · Page 6Linked to original sources
PubMed1996

The DNA polymerase-encoding gene from a thermoacidophilic archaeon Sulfolobus acidocaldarius.

We have cloned, sequenced and characterized the gene encoding a DNA polymerase from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sac). The putative transcription promoter and terminator elements, as well as a potential ribosome-binding site (rbs), have been identified in the flanking regions. One large open reading frame (ORF) found in the sequenced portion of the Sac genome encodes a protein of 875 amino acids (aa). All conserved motifs characteristic of family B of DNA polymerases have been found in the deduced primary structure of this enzyme. The Sac DNA polymerase also contains sequence motifs that form a proofreading exonuclease domain.

Amino Acid Sequence↗
PubMed1996

Cortexillins, major determinants of cell shape and size, are actin-bundling proteins with a parallel coiled-coil tail.

Cortexillins I and II of D. discoideum constitute a novel subfamily of proteins with actin-binding sites of the alpha-actinin/spectrin type. The C-terminal halves of these dimeric proteins contain a heptad repeat domain by which the two subunits are joined to form a two-stranded, parallel coiled coil, giving rise to a 19 nm tail. The N-terminal domains that encompass a consensus actin-binding sequence are folded into globular heads. Cortexillin-linked actin filaments form preferentially anti-parallel bundles that associate into meshworks. Both cortexillins are enriched in the cortex of locomoting cells, primarily at the anterior and posterior ends. Elimination of the two isoforms by gene disruption gives rise to large, flattened cells with rugged boundaries, portions of which are often connected by thin cytoplasmic bridges. The double-mutant cells are multinucleate owing to a severe impairment of cytokinesis.

Actins↗
PubMed1996

A novel class of small amphipathic peptides affect aerial hyphal growth and surface hydrophobicity in Ustilago maydis.

Ustilago maydis, a fungal pathogen of corn, can alternate between yeast-like and filamentous growth. This dimorphic switch is governed by the mating-type loci. We have identified an abundant class of small SDS-insoluble cell wall proteins, designated repellents, specifically present in the filamentous form. Genetic analysis revealed that these peptides are processed from a single precursor protein, Rep1. Rep1 comprises 652 amino acids with a leader sequence for secretion. A characteristic feature of Rep1 is 12 repeats of a 37 amino acid consensus sequence; 10 of these repeats are separated by Kex2 protease cleavage sites. In (delta)rep1 mutants formation of aerial hyphae and surface hydrophobicity were reduced dramatically. This and the fact that expression of rep1 is regulated by the mating-type loci indicates that repellents play a structural role in the formation of aerial hyphae.

Amino Acid Sequence↗
PubMed1996

Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.

SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.

Animals↗
PubMed1996

Enzymatic properties and inhibition by single-stranded autonomously replicating sequences of adenylosuccinate synthase from Saccharomyces cerevisiae.

Adenylosuccinate synthase (ASS) from Saccharomyces cerevisiae has been shown to bind specifically to the T-rich side of the autonomously replicating sequence (ARS) core consensus sequence [Zeidler, R., Hobert, O., Johannes, L., Faulhammer, H. & Krauss, G. (1993) J. Biol. Chem. 268, 20191-20197]. We have cloned and sequenced the gene for ASS and have studied in detail the enzymatic properties and DNA-binding activity of ASS. The deduced amino acid sequence of the yeast ASS is highly similar to the same enzymes from other sources from which it is however distinguished by its more basic nature. We show that the enzymatic activity of ASS is inhibited in a highly specific manner by the binding of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Other nucleic acids, rNTP and dNTP are not able to mimic the specific inhibitory effect. Single-base substitutions in the ARS core sequence lead to a tenfold reduction in inhibition. The inhibition data corroborate the earlier report on the DNA-binding specificity of this enzyme. The homologous enzymes from Escherichia coli and Dictyostelium discoideum do not show specific binding to single-stranded ARS sequences and their enzymatic activity is not influenced by the presence of a 44-base DNA oligonucleotide carrying the ARS core consensus sequence. Treatment of ASS with alkaline phosphatase leads to a loss of DNA binding and to a loss of the inhibition by DNA of the enzymatic activity which suggests that the DNA-binding activity but not the enzymatic activity may be regulated by the phosphorylation status of the protein.

Adenylosuccinate Synthase↗
PubMed1996

A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains.

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.

Amino Acid Sequence↗
PubMed1996

Deletion of a B800-850 light-harvesting complex in Rhodospirillum molischianum DSM119 leads to "revertants" expressing a B800-820 complex: insights into pigment binding.

A B800-850 light-harvesting complex (also called LH2) deficient strain of Rhodospirillum molischianum was constructed by replacing a portion of the LH2 gene cluster by a kanamycin resistance gene cartridge. The LH2 deficient strain was characterized spectroscopically and by Southern blot analysis. Surprisingly, pseudorevertants were obtained which express a B800-820 complex which could not be observed in the wild type. This B800-820 complex was isolated and characterized. It consists of an alpha- and a beta subunit with 56 and 45 amino acid residues, respectively. The amino acid sequences of both subunits are extremely similar to those of the corresponding B800-850 complex. Resonance Raman spectroscopy shows that in the B800-820 complex the two 2-acetylcarbonyl groups of the bacteriochlo-rophyll a (BChl a) molecules absorbing at 820 nm are free from hydrogen bond interactions, whereas one of the two 2-acetylcarbonyl groups of the pair of BChl a molecules absorbing at 850 nm of the B800- 850 complex is involved in hydrogen bonds. These different protein- pigment interactions are due to the replacement of alpha Trp43 in the B800-850 complex by a Phe in the B800- 820 complex. Comparison of the amino acid sequences of the B800-850 and B800-820 complexes of Rs. molischianum and Rhodopseudomonas acidophila reveals a conserved motif comprised of three amino acid residues. Molecular modeling using the known LH2 structure of Rps. acidophila Ac 10050 indicates that this motif might be important for the precise structural arrangement of the native complex and fine tuning of its spectroscopic properties.

Amino Acid Sequence↗
PubMed1996

CMP-N-acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron-sulphur protein to be described in Eukarya.

Electron paramagnetic resonance (EPR) spectroscopy and analysis of the primary structure of the CMP-N-acetylneuraminic acid hydroxylase revealed that this enzyme is the first iron-sulphur protein of the Rieske type to be found in the cytosol of Eukarya. The dithionite-reduced hydroxylase exhibited an EPR signal known to be characteristic for a Rieske iron-sulphur centre (2Fe-2S), the g-values being 1.78, 1.91 and 2.01, respectively. An analysis of the primary structure of the hydroxylase led to the identification of an amino acid sequence, known to be characteristic for Rieske proteins. Furthermore, possible binding sites for cytochrome b5, the substrate CMP-Neu5Ac and a mononuclear iron centre were also identified.

Amino Acid Sequence↗
PubMed1996

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase.

Amino Acid Sequence↗
PubMed1996

Cytoplasmic and mitochondrial forms of yeast adenylate kinase 2 are N-acetylated.

Yeast major adenylate kinase (Aky2p), encoded by a single gene, occurs in two subcellular compartments, mitochondria and cytoplasm. Only 6-8% of the protein which has no cleavable presequence is imported into the organelle (Bandlow et al. (1988) Eur. J. Biochem. 178, 451-457). In the wild type two AKY2-derived signals (a major and a minor one) were detected by a monospecific antibody after two-dimensional gel electrophoresis and Western blotting. The signals reflected identical electrophoretic mobilities and were absent from an AKY2-disrupted strain suggesting that they were due to differently modified forms of Aky2p. Two similar signals were found in a mutant defective in protein N-acetylation, however, the pI values of both spots were shifted towards alkaline pH by one charge. This indicated that both forms of Aky2p were N-acetylated in the wild type and that their charge difference was not caused by incomplete N-acetylation. This observation furthermore suggested that, in the wild type, two different modifications exist one of which is N-acetylation. The second modification remains unidentified. We analysed the influence of protein N-acetylation on mitochondrial import. Both versions of Aky2p occurred in the cytoplasm and in mitochondria. Their proportion was unchanged in the N-acetylation mutant showing that neither modification affected the efficiency of import of adenylate kinase into mitochondria. It is discussed that N-acetylation occurs during or immediately after translation in the cytoplasm so that import of adenylate kinase may ensue co-translationally.

Acetylation↗
PubMed1996

Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.

Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.

Amino Acid Sequence↗
PubMed1996

One-step purification of the beta-glucan elicitor-binding protein from soybean (Glycine max L.) roots and characterization of an anti-peptide antiserum.

A low abundance beta-glucan elicitor-binding protein from soybean was isolated by a rapid, simple and one-step purification method yielding about 9000-fold enrichment. The affinity-based purification technique was more efficient than a procedure that uses conventional methods and preserved the binding activity to a much larger extent. The final preparation consisted of one major protein with an apparent molecular mass of about 75 kDa. Electrophoretic analyses of the purified and photoaffinity-labeled binding protein showed that the native protein was an oligomer with apparent molecular mass of about 240 kDa. A polyclonal anti-peptide antiserum was raised against a synthetic 15-mer internal oligopeptide sequence derived from the 75-kDa protein. The antiserum recognized the purified binding protein in immunoblotting experiments and precipitated the affinity-labeled protein from a crude extract of the membrane fraction.

Animals↗
PubMed1996

Glutathione reductase and glutamate dehydrogenase of Plasmodium falciparum, the causative agent of tropical malaria.

The use of glutathione reductase inhibitors in chemotherapy is the raison d'être for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N-terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first beta strand and the diphosphate-fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P.falciparum glutathione reductase as a drug target are discussed.

Amino Acid Sequence↗
PubMed1996

Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides.

The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.

Amino Acid Sequence↗
PubMed1996

Identification of stress proteins in endothelial cells.

Endothelial cells have been shown to play a major role in the pathophysiology of various diseases including ischemic heart disease and viral infection leading to myocarditis or dilated cardiomyopathy, conditions in which stress proteins (heat shock protein-hsp; glucose-related protein - grp) are likely to be involved. For further characterization of stress proteins and their possible role in these diseases, the major stress proteins in human endothelial cells were separated by two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension and identified by immunoblotting and either N-terminal or internal amino acid sequencing, respectively. Ubiquitin, hsp27, hsp60, hsp70, heat shock cognate protein 70, grp78 and grp75 were found to be constitutively expressed; hsp72 was found in stressed cells, exclusively, in line with results obtained in other human cell lines. Three additional proteins with molecular masses between 34 and 40 were regularly detected in stressed cells that were found to have identical amino acid sequences with those of members of the hsp70 family.

Amino Acid Sequence↗
PubMed1996

Ultraviolet matrix assisted laser desorption ionization-mass spectrometry of electroblotted proteins.

Direct mass spectrometric analysis of proteins electroblotted onto polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is demonstrated by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) with a linear time-of-flight instrument, equipped with a nitrogen laser (337 nm). The blotted proteins were desorbed directly from the blotting membrane after incubation with sinapinic acid as matrix. Different commercially available membranes resulted in high quality protein signals for hydrophobic membranes exhibiting high specific surface areas (Immobilon PSQ or Trans-Blot) or for charged membranes (Immobilon CD). Systematic investigations with standard proteins were performed to compare standard preparation procedures for ultraviolet (UV) MALDI-MS on stainless steel sample stages and preparation of proteins immobilized onto membranes either by direct application from protein solutions (spotting) or by electrotransfer from gels (electroblotting). Aspects such as mass resolution reproducibility from shot to shot and spot to spot, mass accuracy, and preservation of protein localization are addressed in this paper.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Cloning, molecular and functional characterization of Arabidopsis thaliana allene oxide synthase (CYP 74), the first enzyme of the octadecanoid pathway to jasmonates.

Allene oxide synthase, an enzyme of the octadecanoid pathway to jasmonates, was cloned from Arabidopsis thaliana as a full-length cDNA encoding a polypeptide of 517 amino acids with a calculated molecular mass of 58705 Da. From the sequence, an N-terminal transit peptide of 21 amino acids resembling chloroplast transit peptides was deduced. Three out of four invariant amino acid residues of cytochrome P450 heme-binding domains are conserved and properly positioned in the enzyme coding region, including the heme-accepting cysteine (Cys-470). Southern analysis indicated in A. thaliana only one allene oxide synthase gene to be present. While transcript levels were rapidly and transiently induced after wounding of the leaves, allene oxide synthase activity remained nearly constant at a low level of ca. 0.8 nkat per mg of protein. The cDNA encoding A. thaliana allene oxide synthase was highly expressed in bacteria giving rise to a polypeptide of the calculated molecular mass. The protein was enzymatically active, and verification of the reaction products by GC-MS showed that it was capable of utilizing not only 13-hydroperoxylinolenic acid (13-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid), but also 13-hydroperoxylinoleic acid (13-hydroperoxy-9(Z), 11(E)-octadecadienoic acid) as substrate. The data suggest parallel pathways to jasmonates from linolenic acid or linoleic acid in A. thaliana.

Amino Acid Sequence↗