Biomedical subjects

A Bacher

Publications and source records attributed to A Bacher.

At least 163 records · Page 9Linked to original sources

PubMed1992

Crystallization and preliminary crystallographic characterization of GTP cyclohydrolase I from Escherichia coli.

GTP cyclohydrolase I of Escherichia coli has been purified from a recombinant bacterial strain. The enzyme was crystallized from 0.6 M-sodium citrate and from 0.8 M-sodium/potassium phosphate, respectively. Crystals grown in citrate showed X-ray diffraction extending to a resolution better than 3 A. The space group was P2(1) with cell dimensions a = 204.8 A, b = 210.1 A, c = 72.2 A, alpha = gamma = 90 degrees and beta = 95.8 degrees.

Biopterins↗

PubMed1992

Electron microscopy of decorated crystals for the determination of crystallographic rotation and translation parameters in large protein complexes.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.

Bacillus subtilis↗

PubMed1992

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.

Amino Acids↗

PubMed1992

Influence of metabolic inhibitors on the degradation of tight junctions in HT29 cells.

The human colon adenocarcinoma cell line HT 29 grows in culture without tight junctions (TJ). Tight junction strands of the fascia occludens type can be induced by treatment with proteases and are subsequently degraded during a period of about 3 h. Experiments using a variety of metabolic inhibitors such as 2-deoxyglucose, 2,4-dinitrophenol, and CCCP show that the degradation of TJ is retarded under conditions of ATP depletion. Thus it appears that the removal of TJ from the cell surface is an energy-dependent process. Moreover, DNP can specifically inhibit the degradation of TJ even in the absence of ATP depletion. The possible involvement of a proton gradient in the mechanism of TJ degradation is discussed.

2,4-Dinitrophenol↗

PubMed1992

Protease inhibitors suppress the formation of tight junctions in gastrointestinal cell lines.

Tight junctions (TJ) of the fascia occludens type can be induced in the human colon adenocarcinoma cell lines HT29 and Caco-2 by treatment with 320 mM cesium sulfate. This process can be completely inhibited by the protease inhibitors leupeptin and antipain. The concentration for 50% inhibition was 32 microM leupeptin and 270 microM antipain, respectively. In the polarized colon carcinoma cell line Caco-2, the spontaneous formation of histotypical TJ and the development of transepithelial electrical resistance do not occur when the cells are cultured in medium containing 400 microM leupeptin. Following the removal of leupeptin, zonula occludens type TJ and electrical resistance develop synchronously during a period of 4 h. Dihydroleupeptin, the alcohol analog of leupeptin, inhibits neither the spontaneous nor the induced assembly of TJ fibrils. Thus, the aldehyde group of leupeptin is essential for activity. These data suggest that the salt-induced as well as the spontaneous formation of TJ involve cellular proteases which are susceptible to protease inhibitors.

Antipain↗

PubMed1992

Biosynthetic precursors of deazaflavins.

The incorporation of 13C- and 14C-labeled precursors into 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (factor F0) was studied with growing cells of Methanobacterium thermoautotrophicum. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was incorporated into the deazaflavin and into riboflavin without dilution. Tyrosine and 4-hydroxyphenylpyruvate were incorporated into the deazaflavin and into cellular protein. 4-Hydroxybenzaldehyde was not incorporated. A reaction mechanism is proposed for the formation of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine or 4-hydroxyphenylpyruvate.

Flavins↗

PubMed1992

Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.

Amino Acid Sequence↗

PubMed1991

Biosynthesis of 5-hydroxybenzimidazolylcobamid (factor III) in Methanobacterium thermoautotrophicum.

Cultures of Methanobacterium thermoautotrophicum were supplemented with 13C-labeled acetate or pyruvate, and the labeling pattern of the corrinoid, factor III, was established by 13C NMR spectroscopy. Complete 13C signal assignments were obtained by two-dimensional NMR experiments. The labeling pattern of factor III was analyzed by comparison with those of amino acids and nucleosides. The corrin ring system is derived from eight molecules of glutamate. The aminopropanol moiety is derived in a hitherto unknown pathway from pyruvate by reductive amination. The heterocyclic ring of hydroxybenzimidazole shares the labeling pattern of the imidazole ring of purines. The remaining four carbon atoms of the carbocyclic ring show the labeling signature of a carbohydrate with two of the carbons introduced from acetate and two from C-1 of pyruvate. However, erythrose can be ruled out as the specific precursor on the basis of a detailed investigation of aromatic amino acids indicating that erythrose 4-phosphate is obtained by reductive carboxylation of a triose precursor and not by the pentose phosphate cycle.

Acetates↗

PubMed1991

Biosynthesis of riboflavin. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase.

The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed.

Candida↗

PubMed1991

Correlation of metal decoration and topochemistry on protein surfaces.

On the surface of protein molecules the formation of metal clusters during vacuum condensation is controlled by topochemical features of the substrate and by specific properties of the decorating material. The resulting metal distribution (decoration pattern) can be mapped by electron microscopy in conjunction with image processing. We have applied this technique to freeze-etched crystals of the lumazine synthase-riboflavin synthase complex and its derivative obtained by binding of the heteropolytungstate (NaP5W30O110)(NH4)14.31 H2O. The decoration pattern of the free protein and its heteropolytungstate derivative showed marked differences. The correlation of these data with the X-ray structure of the protein showed an increased affinity of both gold and silver to the location of heteropolytungstate. Decoration sites can, but do not need to, be close to the protein surface. Actually, two of the observed decoration sites are located on a layer of ice as thick as 20 A, which apparently transmits underlying topochemical features. Preferential affinity of a site to a given metal must be seen as a property that depends on specific interaction with the decorating material but also on the differential affinities in adjacent areas.

Bacillus subtilis↗

PubMed1991

Biosynthesis of nucleotides, flavins, and deazaflavins in Methanobacterium thermoautotrophicum.

The biosynthesis of deazaflavins, flavins, ribonucleotides, and selected amino acids was studied in Methanobacterium thermoautotrophicum by incorporation of 13C-labeled acetate and pyruvate. 13C enrichments were monitored by 13C and 1H NMR spectroscopy. The biosynthesis of ribonucleotides follows the standard pathway. The xylene ring of riboflavin is formed from two pentose moieties in agreement with studies in yeasts and eubacteria. The pyrimidine ring and the ribityl side chain of the deazaflavin chromophore of coenzyme F420 are derived from the purine nucleotide pool. The phenolic ring and C-5 of the deazaflavin system are supplied by the shikimate pathway. A hypothetical mechanism for the assembly of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione and 4-hydroxyphenyl-pyruvate is proposed.

Amino Acids↗

PubMed1991

15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger.

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.

Aspergillus niger↗

PubMed1991

New aspects of the morphology and function of the human hip joint ligaments.

The capsular ligaments of the human hip joint were submitted to exact morphological analysis, and they proved to be multiple and numerous. We have described various ligamentous systems and their interconnections, and have suggested new terminologies and systematics. The ligaments were subjected to functional analysis by means of measuring strips to determine the positions in which the ligaments are taut. The ligament systems were all found to serve a restrictive function, and various parts of the apparatus restricted all possible movements in the hip joint. Extension is restricted by the medial iliofemoral complex, abduction by the pubofemoral ligament, and adduction by the posterior coxal ligaments and by the superior ischiofemoral ligament. Flexion is restricted by the inferior ischiofemoral ligaments, inward rotation by the superior ischiofemoral ligament, and outward rotation by the lateral iliofemoral complex. Only the ligament of the femoral head is unable to exert a restricting function, despite reaching a state of tension in extreme adduction.

Femur Head↗

PubMed1991

Biosynthesis of tetrahydrofolate. Sequence of GTP cyclohydrolase I from Escherichia coli.

The sequence of the gene coding for GTP cyclohydrolase I of Escherichia coli and of the adjacent regions was determined. The open reading frame contains 669 nucleotides. The deduced amino-acid sequence represents a protein consisting of 223 amino-acid residues with a molecular mass of 24,873 Da. Partial amino-acid sequences of the N-terminal region and of 5 peptides obtained by trypsin and BrCN cleavage were determined by Edman degradation and were in full agreement with the sequence deduced from the nucleotide sequence. The starting methionine is removed by posttranslational modification. The protein shows extensive homology to the recently reported GTP cyclohydrolase from rats.

Amino Acid Sequence↗

PubMed1990

Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase.

The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity.

Candida↗

PubMed1990

Control of tetrahydrobiopterin synthesis in T lymphocytes by synergistic action of interferon-gamma and interleukin-2.

The control of (6R)-5,6,7,8-tetrahydrobiopterin (H4biopterin) synthesis in primed T cells was analyzed by using the human T cell leukemia virus type I (HTLV-I)-transformed T cell line MT-2. In contrast to the slowly progressing induction of H4biopterin synthesis during activation of resting T cells, it is completed during a 59-h period and is directed by a synergism of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Both GTP cyclohydrolase and (6R)-(1',2'-dioxopropyl)-5,6,7,8-tetrahydropterin synthase activities are induced by IFN-gamma. They are further enhanced by combined treatment with IL-2, which per se is ineffective. Furthermore, the combined treatment synchronizes the time periods of both maximum activities, now extending from 33 to 44 h. This period correlates with high cellular H4biopterin levels. It is preceded by a fast and transient period of H4biopterin increase which depends on the synergistic action of both IFN-gamma and IL-2. It coincides with a transient increase in sepiapterin reductase activity. In contrast to MT-2 cells, HTLV-I-transformed HUT 102 cells constitutively secrete IFN-gamma and express IFN-gamma mRNA. The accumulation of H4biopterin is suppressed by anti-IFN-gamma polyclonal antibody and correlates with constitutive expression of all H4 biopterin-synthesizing enzymes.

Alcohol Oxidoreductases↗

PubMed1990

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids.

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.

Bacillus subtilis↗

PubMed1990

Riboflavin synthases of Bacillus subtilis. Purification and amino acid sequence of the alpha subunit.

Bacillus subtilis has two different riboflavin synthases characterized by the subunit structures alpha3 (light enzyme) and alpha3beta60 (heavy enzyme). The light enzyme was purified by a novel procedure with increased yield and excellent reproducibility. The proposed trimer structure was confirmed by cross-linking experiments with dimethyl suberimidate. Fragments of alpha subunits were prepared by cleavage with cyanogen bromide, trypsin, protease Lys-C, and Staphylococcus aureus protease V8, respectively. Sequences were determined by automated liquid or gas phase Edman degradation. The complete sequence (202 amino acids) was established by direct sequencing of the N terminus and sequencing of overlapping peptides. The sequence shows marked internal homology between the NH2-terminal and COOH-terminal half encompassing 26 identical positions and 23 conservative replacements. This suggests that the protomer forms two structurally similar domains. Since it is known that the enzyme has two binding sites per subunit for the substrate 6,7-dimethyl-8-ribityllumazine, it appears likely that each of the homologous protein domains provides one binding site. The stereochemical features of the enzyme mechanism and the structural relation of the alpha trimer to the beta60 capsid of heavy riboflavin synthase suggest that the six domains corresponding to the alpha subunit trimer are related by pseudo 32 symmetry.

Amino Acid Sequence↗