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F Lottspeich

Publications and source records attributed to F Lottspeich.

At least 163 records · Page 9Linked to original sources
PubMed1995

Debranching of arabinoxylan: properties of the thermoactive recombinant alpha-L-arabinofuranosidase from Clostridium stercorarium (ArfB).

The gene arfB encoding alpha-L-arabinofuranosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment. The recombinant gene product ArfB was purified by fast-performance liquid chromatography. It has a tetrameric structure with a monomeric relative molecular mass of 5200. The optima for temperature and pH are 70 degrees C and 5.0 respectively. The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents. It hydrolyzes aryl and alkyl alpha-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism. The identify of the N-terminal amino acid sequences indicates that ArfB corresponds to the major alpha-arabinosidase activity present in the culture supernatant of C. stercorarium.

Amino Acid Sequence↗
PubMed1995

Characterization of relevant membrane antigens by two-dimensional immunoblot and n-terminal sequence analysis in patients with myocarditis.

In myocarditis an antigen-specific immune response to cardiac epitopes has been demonstrated by several investigators. In 25 patients with histologically proven myocarditis, autoantibodies to cardiac tissue were observed in 65% by immunofluorescence assay. By immunoblot analysis 46% of sera reacted with cardiac tissue. These antibodies were preferentially directed against proteins with molecular weights of 43 and 67 kDa. Many of these antibodies have been demonstrated to be cytolytic to isolated cardiocytes in vitro. However, their corresponding antigens on heart muscle cells still remains unclear. 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 in the range of 30-67 kDa at isoelectric points of 6.5-8.5 to be of particular interest. Five of these proteins were identified by immunoblot analysis and two were identified by n-terminal sequence analysis (Edman Degradation) as creatine kinase and a sarcomeric isoenzyme. As creatine kinase is an intracellularly located enzyme, the pathogenetic role of the autoantibodies directed against it remains to be elucidated further.

Adult↗
PubMed1995

Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE.

Homopolymeric alpha-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5' end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.

Amino Acid Sequence↗
PubMed1995

An epitope of elongation factor Tu is widely distributed within the bacterial and archaeal domains.

A monoclonal antibody (MAb), MAb 900, which detects a 43-kDa protein present on Escherichia coli was found. Subsequently, more than 90 organisms, belonging to either the bacterial, archaeal, or eucaryal domain, were tested for reactivity to this MAb. Of the bacterial and archaeal domains, almost all species proved to be positive, whereas all organisms from the eucaryal domain gave negative results. The 43-kDa protein was purified by affinity chromatography and subsequently analyzed by microsequencing methods. Two peptide sequences which showed a high degree of homology (> 99%) to the prokaryotic elongation factor Tu (EF-Tu) were obtained. Western blot (immunoblot) analysis using both purified EF-Tu and EF-Tu domains confirmed that the unknown protein was EF-Tu. The panbacterial distribution of EF-Tu, which is present in large amounts in every prokaryotic cell, renders this protein a good candidate for a diagnostic approach. In consequence, we have used the anti-EF-Tu MAb 900 to design both a dot blot assay and an enzyme-linked immunosorbent assay. From either blood culture, urine, or gall-bladder fluid, bacterial contamination could be detected. The sensitivity of these tests is currently 10(4) bacteria per ml.

Amino Acid Sequence↗
PubMed1995

Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution.

Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.

Allosteric Regulation↗
PubMed1995

The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.

Amino Acid Sequence↗
PubMed1995

A competitive chromogenic assay to study the functional interaction of urokinase-type plasminogen activator with its receptor.

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.

Amino Acid Sequence↗
PubMed1995

Limited proteolysis of alpha 1-proteinase inhibitor (alpha 1-PI) in acute leukemia: studies on the resulting fragments and implication for the structure of the inactivated inhibitor.

In patients with acute myeloid leukemia, a 41 kDa glycoprotein appears in the urine during remission induction chemotherapy. We have recently reported on the isolation and preliminary characterization of this protein and the generation of specific monoclonal antibodies which showed that it is a proteolytically modified form of alpha 1-proteinase inhibitor (Dengler et al., 1992, Biol. Chem. Hoppe-Seyler 373, 581-588). In the paper presented here, results from further characterization experiments as well as from studies on the effects of proteolysis on the conformation and the resulting functional properties of the truncated inhibitor are reported. N-terminal amino acid sequence analysis showed that proteolysis has occurred in the N-terminal part as well as in the reactive site loop of alpha 1-PI. The resulting core protein of 41 kDa is composed of approximately 324 amino acid residues with the C-terminus located close to Lys343 of alpha 1-PI.A 4 kDa peptide remaining bound to this fragment throughout the entire purification procedure could be separated by SDS treatment. N- and C-terminal sequence analysis of this peptide after isolation by gel filtration showed that it is comprised of residues Ile359 up to Lys394, thus representing the peptide located C-terminal to the reactive site loop of alpha 1-PI. Transverse urea gradient gel electrophoresis indicates that the proteolyzed inhibitor is in the thermodynamically stable, relaxed (R)-conformation known for proteinase-complexed and cleaved serpins. The truncated inhibitor exhibits no chemotactic activity towards neutrophils when tested in a standard assay.

Amino Acid Sequence↗
PubMed1995

Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs).

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.

Amino Acid Sequence↗
PubMed1994

Neurotrophin-6 is a new member of the nerve growth factor family.

During vertebrate development, many neurons depend for survival and differentiation on their target cells. The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF). NGF and the other known members of the neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) are conserved as distinct genes over large evolutionary distances. Here we report the cloning of neurotrophin-6 (NT-6), a new member of this family from the teleost fish Xiphophorus. NT-6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT-6 from cell surface and extracellular matrix molecules. Recombinant purified NT-6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT-6 is expressed in the embryonic valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecules may modulate its action in the nervous system.

Amino Acid Sequence↗
PubMed1994

Purification of the synaptic vesicle-binding protein physophilin. Identification as 39-kDa subunit of the vacuolar H(+)-ATPase.

Physophilin is a 36-kDa polypeptide originally identified in synaptic plasma membrane fractions, which binds to synaptic vesicles and has been implicated in vesicle docking and/or exocytosis during neurotransmitter release. Here we report on the purification, amino acid sequence analysis, and subcellular localization of physophilin. Physophilin was enriched from detergent extracts of crude synaptic plasma membranes by a combination of cation exchange and lentil-lectin chromatography. Sequence analysis of peptides generated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that physophilin is identical to the 39-kDa subunit (Ac39) of the vacuolar H(+)-ATPase. This was confirmed further by Western blot analysis with an Ac39-specific antiserum and by vesicle binding assays with recombinant Ac39 protein. Subcellular fractionation showed that Ac39 is enriched in synaptic vesicles, with lesser amounts being present in synaptic plasma membrane fractions. These results argue against a docking role of physophilin/Ac39 in synaptic vesicle exocytosis.

Amino Acid Sequence↗
PubMed1994

Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.

Base Sequence↗
PubMed1994

Cloning of a novel malignant melanoma-derived growth-regulatory protein, MIA.

Growth and progression of malignant melanoma cells is influenced by a complex network of growth-stimulating and -inhibiting factors produced by both the tumor cells and the local environment. Here we report the purification and molecular cloning of a novel growth regulating protein, designated melanoma inhibitory activity (MIA) and provide a preliminary functional characterization. MIA is translated as a 131-amino acid precursor and processed into a mature 107-amino acid protein after cleavage of a putative secretion signal. A murine complementary DNA was isolated that encoded a MIA-protein with 88% amino acid identity. MIA is secreted into the culture supernatant by several malignant melanoma cell lines as an M(r) 11,000 autocrine growth factor and acts as a potent tumor cell growth inhibitor for malignant melanoma cells and some other neuroectodermal tumors, including gliomas. MIA has no homology to any other known protein and, therefore, represents a novel type of growth-regulatory factor. Furthermore, we describe a molecular approach to express functionally active MIA in Escherichia coli, which might be attractive as a future antitumor therapeutical substance.

Amino Acid Sequence↗
PubMed1994

The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved.

The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.

Anaerobiosis↗
PubMed1994

A novel mediator of class II gene transcription with homology to viral immediate-early transcriptional regulators.

Our investigations of mammalian class II gene transcription resulted in identification, purification, and cloning of the corresponding cDNA of a cellular factor (p15) that mediates the effects of several distinct activators on transcription in vitro. Functional deletion analyses revealed a bipartite structure of p15 comprising an amino-terminal regulatory domain and a carboxy-terminal cryptic DNA-binding domain. We provide evidence that activity of p15 is controlled by protein kinases that target the regulatory domain. Structural and functional similarities, including sequence homology to domains essential for cofactor function, cofactor activity, promiscuity with respect to transcriptional activators, and interactions with components of the basal transcription machinery, relate this novel cellular cofactor to viral immediate-early transcriptional regulators.

Amino Acid Sequence↗
PubMed1994

Isolation and molecular cloning of prostacyclin synthase from bovine endothelial cells.

Prostacyclin synthase catalyzes the conversion of prostaglandin H2 to prostacyclin, which is a powerful vasodilator and the most potent natural occurring inhibitor of platelet aggregation. In the present study, we determined the amino acid sequence of bovine prostacyclin synthase by combined protein chemical and molecular cloning techniques. The enzyme was purified and characterized from bovine aorta microsomes, and the partial amino acid sequences were determined with the native enzyme and endoproteinase Lys-C-cleaved peptides. Using primers synthesized according to the amino acid sequences, cDNA coding for prostacyclin synthase was amplified by polymerase chain reaction with bovine endothelial cell poly(A)+ RNA and cloned into pBluescript II. Nucleotide sequence analyses of the cloned cDNA inserts revealed that cDNA for this enzyme contained a 1500-base pair open reading frame coding for a 500-amino acid polypeptide with a M(r) of 56,628. COS-7 cells transfected with an expression plasmid harboring this cDNA clone expressed prostacyclin synthase activity. The primary structure of the enzyme showed structural characteristics of cytochrome P450 and exhibited a 32% identity to that of human cholesterol 7 alpha-hydroxylase. However, the identity between the amino acid sequences of bovine prostacyclin synthase and human thromboxane synthase was only 16%, and no P450 showed an identity higher than 40%, suggesting that prostacyclin synthase represents a new family in the P450 superfamily. RNA blot analysis indicated that the mRNA for prostacyclin synthase from bovine endothelial cells showed a size of approximately 2.7 kilobases and that the mRNA level increased about 3-fold by treatment of tumor necrosis factor-alpha.

Amino Acid Sequence↗
PubMed1994

Existence of a molecular ruler in proteasomes suggested by analysis of degradation products.

Analysis of the degradation products from two proteins, the insulin B-chain and human hemoglobin, generated by archaebacterial Thermoplasma acidophilum 20 S proteasomes, revealed an unexpectedly broad specificity. In spite of the vast number of different peptides found, they fell into a rather narrow size range. This suggests that a molecular ruler exists which determines the length of the cleavage products.

Amino Acid Sequence↗