Biomedical subjects

F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 199 records · Page 11Linked to original sources

PubMed1994

The myelin protein CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase): immunoaffinity purification of CNP from pig and rat brain using a monoclonal antibody and phosphorylation of CNP by cyclic nucleotide-dependent protein kinases.

A monoclonal antibody (MAb-46-1) specifically recognizing a 46 kDa basic protein solubilized from brain membranes was used to prepare an affinity column, which allowed a one-step purification of the 46 kDa protein to homogeneity starting from solubilized cerebellar membranes. MAb-46-1 could also immunoprecipitate the 46 kDa protein from solubilized pig or rat cerebellar membranes. Microsequence analysis of affinity purified 46 kDa protein treated with Lys C demonstrated the identity of the 46 kDa protein as a myelin associated protein, i.e. 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37). The amino acid sequences obtained for the porcine CNP were nearly identical with the known sequences of the bovine and human isoforms but only partially with those of rat and mouse CNP. In SDS PAGE the porcine CNP appeared as a doublet of 44.6 and 45.9 kDa. Both bands of the doublet were equally well recognized by MAb-46-1. Porcine CNP was rapidly and specifically phosphorylated by both protein kinase A and cGMP-dependent protein kinase.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

Molecular characterization of the p100 gene of Borrelia burgdorferi strain PKo.

The p100 gene coding for the p100 protein of Borrelia burgdorferi strain PKo has been cloned, sequenced and expressed in Escherichia coli. An open reading frame including upstream and downstream sequences with potential translation and transcription signals could be identified. The reading frame consists of 1989 nucleotides corresponding to a protein of 663 amino acids and a calculated molecular mass of 75.8 kDa. The protein has a leader peptide and is processed without modification at the N-terminus. A high percentage of amino acid sequence identity could be found to the high-molecular mass protein p83/p93 of B. burgdorferi strain B31.

Amino Acid Sequence↗

PubMed1993

Determination and mutational analysis of the phosphorylation site in the hypusine-containing protein Hyp2p.

Electrospray mass spectrometry of the purified isoforms of the hypusine-containing protein of Saccharomyces cerevisiae Hyp2p suggested a phosphorylation of the acidic isoform, which was confirmed by phosphatase treatment. The phosphorylation site was mapped to the N-acetylated serine residue in position no. 1 by mass spectrometric analysis of enzymatic fragments. Mutation of this serine residue gives rise to only the basic isoform, confirming our protein chemical data. As this mutation has no effect on cell viability or growth rate, the unphosphorylated isoform is sufficient to exert the essential in vivo function of Hyp2p.

Amino Acid Sequence↗

PubMed1993

The DNA-dependent RNA-polymerase of Thermotoga maritima; characterisation of the enzyme and the DNA-sequence of the genes for the large subunits.

An improved purification procedure for Thermotoga maritima RNA-polymerase holoenzyme was developed. The enzyme is highly active with poly dAT or T7 phage DNA as template. DNA gyrase was found to be a side product of this RNA-polymerase purification. The genes for the large subunits beta and beta' of RNA-polymerase were cloned and sequenced. The phylogenetic position of T.maritima within the bacterial domain was determined by various methods. It is the lowest bacterial offspring but slightly higher than the chloroplasts.

Amino Acid Sequence↗

PubMed1993

Yeast coatomer contains a subunit homologous to mammalian beta'-COP.

The homologue of the mammalian coatomer complex was isolated from yeast cytosol and separated on a modified urea-containing SDS-polyacrylamide gel system. An additional band in the 100 kDa molecular weight range appeared when compared to the protein pattern obtained in conventional Laemmli gels, exactly as observed for mammalian coatomer. Cross-reactivity with an anti-peptide antibody raised against the C-terminus of beta'-COP from bovine, and N-terminal sequence analysis, revealed that this protein from yeast is related to beta'-COP from mammals.

Amino Acid Sequence↗

PubMed1993

High molecular weight aspartic endopeptidase generates a coronaro-constrictory peptide from the beta-chain of hemoglobin.

Studying the influence of brain cathepsin D (EC 3.4.23.5) and high molecular weight (HMW) aspartic endopeptidase (EC 3.4.23.-) on the processing of hypothalamic calmodulin-binding coronaro-constrictory peptide factors from the beta-chain of globin it was found that only HMW aspartic endopeptidase generates the fragment 31-40 of the beta-chain of bovine hemoglobin (Hb) by cleavage of the Leu30-Leu31 and Phe40-Phe41 bonds. Digestion of the beta-chain of globin was performed at 37 degrees C at an enzyme/substrate ratio of 1:80 at pH 3.5 using different times of incubation (from 4 h to 10 h). The resulting peptides were separated by reversed-phase high-performance liquid chromatography (HPLC) and then identified by amino acid analysis and Edman degradation. The differences in specificity and activity of these two brain aspartic proteinases could be explained by their different structural features. Our finding provides evidence for a different biological function of these two enzymes. Data obtained give us reason to suppose that HMW aspartic proteinase probably can participate in the processing of the coronaro-constrictory peptide in vivo by limited proteolysis of Hb or Hb-like protein.

Amino Acid Sequence↗

PubMed1993

The 100 kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype ('protovillin').

The 100 kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133-144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions. (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution. (ii) The villin-like molecule in Dictyostelium appears to be a premature villin ('protovillin') which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein.

Actin Depolymerizing Factors↗

PubMed1993

Subunit composition, crystallization and preliminary crystallographic studies of the Desulfovibrio gigas aldehyde oxidoreductase containing molybdenum and [2Fe-2S] centers.

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDS/PAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at 4 degrees C, pH 7.2, using isopropanol and MgCl2 as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6(1)22 or its enantiomorph, with cell dimensions a = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 x 10(-3) nm3/Da (2.5 A3/Da), consistent with the experimental crystal density, rho = 1.14 g/cm3. One dimer (approximately 2 x 100 kDa) is located on a crystallographic twofold axis.

Aldehyde Oxidoreductases↗

PubMed1993

Nucleotide sequence of a putative succinate dehydrogenase operon in Thermoplasma acidophilum.

28 amino acids from the N-terminal region of a putative terminal oxidase from the archaebacterium Thermoplasma acidophilum were determined by Edman degradation. On basis of this amino acid sequence a degenerated oligonucleotide was synthesized and used as a radioactive probe for Southern blot analysis of EcoRI digested genomic DNA. A 2.3 kb EcoRI fragment strongly hybridized to the probe and size selected genomic library from genomic DNA was constructed. Several clones scored positive by screening the library with the degenerated oligonucleotide, from which only one clone contained a EcoRI DNA fragment encoding the 28 amino acid sequence determined by protein sequencing. Sequence analysis revealed the presence of three genes in the typical arrangement of an operon. The first gene codes for a protein containing 11 cystein residues in an arrangement typical for Fe/S proteins. Protein sequence comparison revealed significant homologies to the fumarate reductase and succinate dehydrogenase of other bacteria. The two other genes encode small hydrophobic proteins probably serving as membrane anchor for the Thermoplasma acidophilum succinate dehydrogenase.

Amino Acid Sequence↗

PubMed1993

Ubiquitin found in the archaebacterium Thermoplasma acidophilum.

Systematic N-terminal sequencing of the low molecular weight proteins from Thermoplasma acidophilum separated by two-dimensional poly-acrylamide gel electrophoresis led to the discovery of a polypeptide with an apparent M(r) of 4.5 kDa identical as its first 18 amino acid residues to human ubiquitin. The occurrence of ubiquitin and proteasomes in an archaebacterium strongly suggests that ATP-ubiquitin-dependent proteolysis is a cellular function that developed early in evolution.

Amino Acid Sequence↗

PubMed1993

How Y chromosomes become genetically inert.

We have investigated the mechanistic aspects of inactivation of the major larval cuticle protein genes (Lcp1-4) in Drosophila miranda during Y chromosome evolution. The Lcp genes are located on the X2 and neo-Y chromosomes in D. miranda but are autosomally inherited in all other Drosophila species investigated so far. In the neo-Y chromosome all four Lcp loci are embedded within a dense cluster of transposable elements. The X2 Lcp1-4 loci are expressed, while the Y chromosomal Lcp3 locus shows only reduced activity and the Lcp1, Lcp2, and Lcp4 are completely inactive. Our results suggest that Lcp1 and Lcp3 loci on the degenerating Y chromosome of D. miranda are silenced by neighboring transposable elements. These observations support our assumption that the first step in Y chromosome degeneration is the successive silencing of Y chromosomal loci caused by trapping and accumulation of transposons.

Amino Acid Sequence↗

PubMed1993

Unexpected similarities of the B800-850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria.

The B800-850 light-harvesting complex (also called LH2) was isolated from photosynthetic membranes of Rhodospirillum molischianum DSM 119 using molecular sieve and ion-exchange chromatography. Its two bacteriochlorophyll a-binding polypeptides (alpha-subunit and beta-subunit) were purified with a reverse-phase HPLC system. The complete amino acid sequences of both subunits have been determined. The alpha- and beta-subunits consist of 56 and 45 amino acids, respectively, corresponding to molecular weights of 5939 and 5133. In contrast to the B800-850 complexes from other photosynthetic bacteria, the native B800-850 complex from Rs. molischianum is most likely an octamer of monomers with a stoichiometry of three bacteriochlorophyll a and 1.5 lycopenes per alpha,beta-subunit. Resonance Raman spectra provide evidence for a 5-coordinated Mg2+ in the BChl, and a carotenoid mainly in the all-trans configuration. A comparison between resonance Raman data from different photosynthetic bacteria indicates that the BChl a-binding site of the B800-850 complex from Rs. molischianum is more similar to the B870 complexes (also called LH1) than to the B800-850 complexes of other photosynthetic bacteria. Sequence similarities especially between the beta-subunits of the B800-850 complex of Rs. molischianum and the B870 and B800-850 complexes of other photosynthetic bacteria agree with this result and provide information on the mode of pigment binding in bacterial antenna complexes.

Amino Acid Sequence↗

PubMed1993

Coactosin, a 17 kDa F-actin binding protein from Dictyostelium discoideum.

A 17 kDa protein, designated as coactosin, has been purified from an actin-myosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F-actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent-insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA-derived amino-acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin-binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins.

Actomyosin↗

PubMed1993

Structural characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins.

Various blotting membranes were evaluated and correlated with the efficiency of electroblotting and the performance in the sequencing process. Structural parameters including specific surface area, pore size distribution, pore volumes, and permeabilities of different solvents lead to discrimination of the membranes relative to their accessible surfaces and membrane densities. Protein binding capacities as well as protein recoveries in electroblotting correlate with the specific surface areas. Almost quantitative retention of proteins during electroblotting from gels was obtained for membranes with a high specific surface area and narrow pores (Trans-Blot, Immobilon PSQ, Fluorotrans), whereas membranes with a relatively low specific surface area (Immobilon P, Glassybond) showed reduced recoveries of between 10-20% for the tested proteins. Initial yields and repetitive yields were compared for radioiodinated standard proteins that have been either electroblotted or loaded by direct adsorption. The results showed that the different permeabilities for solutions of the Edman chemistry have a major influence on initial yields. The glass fiber-based membranes with an extremely low flow restriction produce consistently high initial yields independent of the application mode of the protein (spotted or electroblotted) or the application of the membranes into the cartridge (discs or small pieces). In contrast, the polymeric membranes showed decreasing initial yields with increasing membrane density for spotted and electroblotted proteins. Yields varied considerably when the membranes were applied as discs into the cartridge. This effect could be minimized by cutting the membranes into pieces as small as possible, as demonstrated for electroblotted proteins.

Electrophoresis↗

PubMed1993

beta'-COP, a novel subunit of coatomer.

Several lines of evidence favour the hypothesis that intracellular biosynthetic protein transport in eukaryotes is mediated by non-clathrin-coated vesicles (for a review see Rothman and Orci, 1992). The vesicles have been isolated and a set of their surface proteins has been characterized as coat proteins (COPs). These COPs exist in the cytosol as a preformed complex, the coatomer, which was prior to this study known to contain six subunits: four (alpha-, beta-, gamma- and delta-COP) with molecular weights between 160 and 58 kDa, and two additional proteins of approximately 36 and 20 kDa, epsilon- and xi-COP. Here we describe a novel subunit of the coatomer complex, beta'-COP. This subunit occurs in amounts stoichiometric to the established COPs both in the coatomer and in nonclathrin-coated vesicles and shows homology to the beta-subunits of trimeric G proteins.

Amino Acid Sequence↗

PubMed1993

The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34.

During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. To investigate the protein-protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N-terminal 42 kDa ATPase region and the C-terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

Molecular cloning, sequence analysis and elicitor-/ozone-induced accumulation of cinnamyl alcohol dehydrogenase from Norway spruce (Picea abies L.).

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated M(r) of 38,777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

Alcohol Oxidoreductases↗

PubMed1993

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants.

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.

Aerobiosis↗