bluuebunny/crossref_metadata_2025 huggingface dataset:
A High‐Potential Soluble Cytochrome C ‐551 from the Purple Phototrophic Bacterium Chromatium vinosum is Homologous to Cytochrome C8 from Denitrifying Pseudomonads
Authors: Samyn Bart , De Smet Lina , Van Driessche Gonzalez , Meyer Terrance E. , Bartsch Robert G. , Cusanovich Michael A. , Van Beeumen Jozef J.
Published: August 2004

A minor cytochrome c‐551 component of Chromatium vinosum was previously found to efficiently couple electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center. We have now determined the amino acid sequence of this cytochrome c ‐551 and find that it is homologous to cytochrome c8 (formerly called Pseudomonas cytochrome c ‐551). It is most similar to Methylophilus methylotrophus, Rhodocyclus tenuis, and Azotobacter vinelandii cytochromes c8 (respectively, 57%, 52% and 51%). The C. vinosum cytochrome c, has a single residue insertion relative to Pseudomonas and Azotobacter cytochromes c8. It has fewer charged residues than its homologs and is essentially neutral, which may explain why it is less soluble than the others. The cytochromes c8 are only very distantly related to the cytochromes c2 found in other species of purple bacteria which are much larger in size and which usually mediate electron transfer between the cytochrome bc1 complex and the reaction center. The photosynthetic pathway in Chromatium thus appears to be radically different from that in purple non‐sulfur bacteria.
Characterization of the Gene Encoding Quinohaemoprotein Ethanol Dehydrogenase of Comamonas testosteroni
Authors: Stoorvogel Joke , Kraayveld Diana E. , Van Sluis Cees A. , Jongejan Jaap A. , De Vries Simon , Duine Johannis A.
Published: August 2004

The gene encoding quinohacmoprotein ethanol dehydrogenase type I (QH‐EDH1) from Comamonas testnsteroni has been cloned and sequenced. Comparison of the amino acid sequence deduced from this with that determined for the N‐terminal amino acid stretch of purified QH‐EDH1, suggests that the gene also contains a leader sequence of 31 residues. Based on this information, the molecular mass of the apo‐enzyme, i.e. the enzyme without the cofactors pyrroloquinoline quinone (PQQ) and haem c, and without the C2+, appears to be 73200 Da. Alignment of the deduced amino acid sequence to that of other PQQ‐containing dehydrogenases showed that good similarity (up to 43% identity) exists with most of them. This also showed that the amino acid residues presumed to he involved in PQQ and Ca2+ binding and in the typical features of structure and catalysis of methanol dehydrogenase, are conserved at the same positions in QH‐EDHI, The C‐terminal part of the protein, containing the haem c, exhibited some similarity to cytochromes c553 from cyanobacteria and algae. Correct processing of the qhedh gene appeared lo occur in Escherichia coli strain JM 109 in which the gene was placed under control of the lac promoter, as judged from a positive reaction with antibodies raised against authentic QH‐EDH1, the size of the protein, the presence of haem c in it, and the specific activity value obtained after reconstitution with PQQ. The qhedh gene seems to form part of an operon which is organized in a way different from that of the genes required for methanol oxidation in methylotrophic bacteria.
Isolation, Characterisation and Crystallisation of a Water‐Soluble Fragment of the Rieske Iron‐Sulfur Protein of Bovine Heart Mitochondrial bc1 Complex
Authors: Link Thomas A. , Saynovits Monica , Assmann Claus , Iwata So , Ohnishi Tomoko , Von Jagow Gebhard
Published: August 2004

A water‐soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe‐2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron‐sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane‐bound iron‐sulfur protein. The redox potential as determined by EPR‐monitored redox titration was +306 mV. The far‐ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant α‐helix content was detected in the membrane anchor of the complete iron‐sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle‐shaped single crystals have been grown by the hanging‐drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple‐wavelength anomalous‐scattering technique is underway.
Comparative Study of the Metabolic Pools of Sphingomyelin and Phosphatidylcholine Sensitive to Tumor Necrosis Factor
Authors: Andrieu Nathalie , Salvayre Robert , Levade Thierry
Published: August 2004

The metabolism and localization of the pools of sphingomyelin and phosphatidylcholine (PtdCho) which are hydrolyzed upon activation of the sphingomyelin signal transduction pathway were studied in human skin fibroblasts treated with tumor necrosis factor α (TNF‐α). In a first series of experiments, cellular phospholipids were labeled with [3H]choline under conditions that inhibit the vesicular traffic to the plasma membrane. Thus, in human fibroblasts metabolically labeled in the presence of brefeldin A, monensin or at 20 °C, the arrival of newly synthesized sphingomyelin to the cell surface was prevented, supporting previous conclusions for a vesicular mechanism of sphingomyelin transport to the plasma membrane. Under these conditions, TNF‐α induced the hydrolysis of PtdCho but did not promote the hydrolysis of 3H‐labeled sphingomyelin, suggesting that the sphingomyelin signaling pool resides in a compartment distal to the Golgi apparatus, and possibly in the plasma membrane. TNF was also unable to trigger the breakdown of a radioactive sphingomyelin, [ceramide ‐3H] sphingomyelin, exogenously added to the cells to label the exoplasmic side of the cell surface. However, TNF caused PtdCho and sphingomyelin degradation in fibroblasts that had been treated with bacterial sphingomyelinase to degrade the sphingomyelin pool of the external leaflet of the plasma membrane. A similar result was obtained at 4°C, i.e. under conditions which inhibit endocytosis, thereby excluding the endosomes as a potential site for TNF‐induced sphingomyelin hydrolysis. Altogether, these results strongly argue for a localization of the sphingomyelin signaling pool at the inner leaflet of the plasma membrane, but neither in the endolysosomal nor the Golgi compartments. In addition, when [3H]choline‐labeled fibroblasts were treated under non‐lytic conditions with bacterial phospholipase C to degrade the external pool of PtdCho, TNF was still able to stimulate the hydrolysis of PtdCho. This demonstrates that the pool of PtdCho involved in TNF‐α signaling (and which is hydrolyzed concurrently with sphingomyelin to generate diacylglycerol), is not located in the outer leaflet of the plasma membrane.
cis‐Acting Elements and Transcription Factors Involved in the Intestinal Specific Expression of the Rat Calbindin‐D9k Gene
Authors: Lambert Mireille , Colnot Sabine , Suh EunRan , L'horset Fabienne , Blin Claudine , Calliot Marie‐Elise , Raymondjean Michel , Thomasset Monique , Traber Peter G. , Perret Christine
Published: August 2004

The calbindin‐D9k (CaBP9k) gene is mainly expressed in differentiated duodenal epithelial cells and is used as a model for studying the molecular mechanisms of intestine‐specific transcription. The gene has been cloned, two major DNase‐I‐hypersensitive sites in the duodenum have been described, and a vitamin‐d‐response element has been identified. We have now analysed the transcription factors and regulatory sequences involved in the transcription of the CaBP9k gene in the intestine in ex vivo and in vitro experiments. Transfection experiments in intestinal (CaCo‐2) and non‐intestinal (HeLa) cell lines defined two regions in the 5′‐flanking sequences of the rat CaBP9k gene. A minimal proximal region (−117 to +20) promoted transcription in both intestinal expressing and non‐expressing cell lines. Tissue specificity was conferred by the sequences situated further upstream, which are responsible for complete repression in the non‐intestinal cells. Intestinal transcription was specified by the proximal region, containing a specialized TATA box, and a distal region, which contains a previously described intestinal DNase‐I‐hypersensitive site. In vitro DNase I footprinting, electrophoretic mobility shift assays and antibody supershift assays were used to examine the factors bound to the proximal promoter region (−800 to +80 bp). Rat duodenal nuclear extracts protected 12 sites. Some of them appear to be binding sites for ubiquitous (nuclear factor 1) or hepatic‐enriched sites (hepatocyte nuclear factors 1 and 4, enhancer binding protein α and β) factors. DNA binding studies and transfection experiments indicated that an intestine‐specific transcription factor, caudal homeobox‐2, binds to the TATA box of the rat CaBP9k gene. These data contribute to our understanding of the control of the intestinal transcription of the CaBP9k gene and demonstrate that several trans‐acting factors, other than the vitamin D receptor, may be factors for intestine‐specific CaBP9k gene expression.
Selective Up‐Regulation of α1‐Chimaerin mRNA in SK‐N‐SH Neuroblastoma Cells by K+‐Induced Depolarisation
Authors: Dong Jing‐Ming , Lim Louis
Published: August 2004

The expression of α1‐chimaerin, which encodes a neuron‐specific GTPase‐activating protein for p21rac, is spatially and temporally regulated in vivo. In vitro, expression of the mRNA of both α1‐chimaerin and its alternative spliced form, α2‐chimaerin, was up‐regulated when human neuroblastoma SK‐N‐SH cells underwent neuronal‐type differentiation in a serum‐free medium. KCl‐induced membrane depolarisation also specifically up‐regulated α1‐chimaerin mRNA expression in SK‐N‐SH cells at the transcriptional level. The up‐regulation of α1‐chimaerin expression by membrane depolarisation is not an immediate early event, and occurs 3 h after KCl treatment. It does not require de novo protein synthesis. The increase in calcium influx via the L‐type voltage‐sensitive calcium channel as the result of depolarisation is a key event leading to the up‐regulation of α1‐chimaerin mRNA. α1‐Chimaerin expression was also found to respond positively to the hypertonic osmolarity changes. These results suggest that in vivo expression of α1‐chimaerin, a potential signal transduction molecule, may be regulated by neuronal/synaptic activity.
Purification and Characterization of the Soluble Interleukin‐6 Receptor from Human Plasma and Identification of An Isoform Generated through Alternative Splicing
Authors: Müller‐newen Gerhard , Köhne Christian , Keul Radovan , Hemmann Ulrike , Müller‐esterl Werner , Wijdenes John , Brakenhoff Just P. J. , Hart Margreet H. L. , Heinrich Peter C.
Published: August 2004

The soluble human interleukin‐6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000‐fold enrichment with a yield of 95% was achieved. A subsequent IL‐6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute‐phase protein α1‐antichymotrypsin in HepG2 cells stably transfected with IL‐6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N‐glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane‐bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity‐purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.
Biochemical and Antibacterial Analysis of Human Wound and Blister Fluid
Authors: Frohm Margareta , Gunne Hans , Bergman Ann‐Charlotte , Agerberth Birgitta , Bergman Tomas , Boman Anita , Lidén Sture , Jörnvall Hans , Boman Hans G.
Published: August 2004

Fluid from a post‐operative wound, six leg ulcers and a large blister were collected and analysed by biochemical, microbiological and immunological techniques. The results were compared with those from sera. All samples were lyophilized and extracted twice with 60% aqueous acetonitrile containing 1% trifluoroacetic acid. The pooled supernatants were lyophilized, redissolved, and the fluid extracts were characterized by six techniques (the blister exudate only with three): reverse‐phase HPLC, Edman degradation, mass spectrometry, Western blot analysis, inhibition zone assay on plates with Bacillus megaterium (anti‐Bm activity) and zone clearing on plates with cell walls from Micrococcus luteus (a lysozyme assay). The material corresponding to HPLC peaks of the wound fluid extract was identified as: histone H2B fragments 1–11,1 – 15 and 1 – 16, intact thymosin β‐4, defensins HNP1, 2 and 3, lysozyme and the peptide antibiotic FALL‐39 and its precursor(s). The HPLC‐separated blister fluid was extremely rich in anti‐Bm activity (mainly defensins) and lysozyme. It may also contain factors not identified before. The plate assays scored 50‐fold differences in anti‐Bm activities and more than 10‐fold differences in lysozyme, factors which together with thymosin could be active in wound healing. It is concluded that analysis of wound fluid yields peptide and activity patterns with novel fragments of important peptides, and quantitative differences, that can be useful to understand molecular mechanisms of wound healing further.
Immunological Detection of Proteins Similar to Bacterial Proteases in Higher Plant Chloroplasts
Authors: Ostersetzer Oren , Tabak Sarit , Yarden Oded , Shapira Roni , Adam Zach
Published: August 2004

Despite numerous demonstrations of protein degradation in chloroplasts of higher plants, little is known about the identity of the proteases involved in these reactions. To identify chloroplast proteases by immunological means, we investigated two proteins: ClpP, a protein similar to the proteolytic subunit of the bacterial ATP‐dependent Clp protease, for which a gene is found in the chloroplast genome [Maurizi, M. R., Clark, W. P., Kim, S. H. & Gottesman, S. (1990) J. Biol. Chem. 265, 12546–12552] and PrcA, a cyanobacterial Ca2+‐stimulated protease [Maldener, I., Lockau, W., Cai, Y. & Wolk, P. (1991) Mol. & Gen. Genet. 225, 113–120]. We expressed the clpP gene from rice in Escherichia coli, purified its product, and generated antibodies against the product. Western blot analysis revealed the ClpP protein in different leaf extracts. Analysis of fractionated barley chloroplasts revealed that the protein was associated with the stromal fraction. The expression of ClpP is light independent and tissue specific, as it was found in green and etiolated barley leaves, but not in roots. A second protein, similar to the cyanobacterial protease PrcA, was also detected in chloroplasts. Antibody against this protease recognized proteins in various leaf extracts. When pea chloroplasts were fractionated, the antibody only recognized a stromal protein. The expression of this protein is regulated by light, as it was found in green leaves but not in etiolated leaves. The tissue specificity of PrcA was similar to that of ClpP in that it could not be detected in root extracts.
Identification, Nuclear Localization, and Binding Activities of OZF, A Human Protein Solely Composed of Zinc‐Finger Motifs
Authors: Ferbus Didier , Le Chalony Catherine , Prospéri Marie‐Thérèse , Muleris Martine , Vincent‐Salomon Anne , Goubin Gérard
Published: August 2004

The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc‐finger motifs which belongs to the Krüppel family of zinc‐finger proteins. The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin. These binding activities are characteristic of zinc‐finger proteins. Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells. which is consistent with the pattern of expression observed at the RNA level in cell cultures. Western blot analysis demonstrated the expression of a 33‐kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans‐acting domain. These data establish the unique structure of the OZF protein which is distinct from previously identified zinc‐finger proteins. In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis.
Chemotactic 5‐Oxo‐Icosatetraenoic Acids Activate A Unique Pattern of Neutrophil Responses
Authors: Norgauer Johannes , Barbisch Michael , Czech Wolfgang , Pareigis Johanna , Schwenk Uwe , Schröder Jens‐Michael
Published: August 2004

Neutrophil cell responses and signal pathways elicted by the chemotactic arachidonic acid metabolites (6E, 8Z, 11Z, 14Z)‐5‐oxo‐icosatetraenoic acid and (6E, 8Z, 11Z, 13E)‐5‐oxo‐15‐hydroxy‐icosatetraenoic acid were studied and compared with those of other chemotaxins. Polyphosphoinositol lipid analysis revealed activation of phosphatidylinositol‐bisphosphate 3‐kinase by both agonists. Experiments with Fura‐2 in the presence of EGTA indicated Ca2+ mobilization from intracellular stores by both 5‐oxo‐icosanoids. A transient actin response and production of small amounts of superoxide anions upon stimulation with both agents was detected. The changes induced by 5‐oxo‐icosanoids were more moderate and transient than those obtained by other chemotaxins. Desensitization studies indicated cross‐desensitization between both 5‐oxo‐icosanoids, but no interference with the response of other chemotaxins. All cell responses elicted by 5‐oxo‐icosanoids were inhibited by pertussis toxin suggesting involvement of G‐proteins, a common activation mechanism for all known potent chemotaxins. In contrast to other chemotaxins, 5‐oxo‐icosanoids at concentrations 500‐fold higher than the ED50 of other functions did not induce up‐regulation of CD11b and N‐formyl‐peptide receptors at the cell surface, and failed to potentiate N‐formyl‐peptide‐induced superoxide anion production. These results indicate that 5‐oxo‐icosanoids trigger a unique pattern of neutrophil responses.
Reconstitution and Pigment‐Binding Properties of Recombinant CP29
Authors: Giuffra Elisabetta , Cugini Daniela , Croce Roberta , Bassi Roberto
Published: August 2004

The minor light‐harvesting chlorophyll‐a/b ‐binding protein CP29 (Lhcb4), overexpressed in Esche‐richia coli, has been reconstituted in vitro with pigments. The recombinant pigment‐protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light‐harvesting chlorophyll‐alb‐binding protein of photosystem II (LHCII).

The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll‐a/b ‐content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146–150; Paulsen, H., Riimler, U. & Riidiger, W. (1990) Planta (Heidelb.) 181, 204–211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.
Characterization of Centrin Genes in Paramecium
Authors: Madeddu Luisa , Klotz Catherine , Le Caer Jean‐Pierre , Beisson Janine
Published: August 2004

Centrins are highly conserved, ubiquitous cytoskeletal components which belong to the EF‐hand superfamily of Ca2+‐modulated proteins. We report here the molecular characterization of new members of the centrin family, Paramecium centrins. Previous studies described the organization of the infraciliary lattice (ICL), the innermost cortical cytoskeletal network of Paramecium, and showed that it was composed of a set of low‐molecular‐mass, Ca2+‐binding polypeptides [Garreau de Loubresse, N., Klotz, C., Vigues, B., Rutin, J. & Beisson, J. (1991) Biol. Cell 71, 217–225]. In this paper we show that these polypeptides are recognized by specific anti‐centrin polyclonal antibodies. Their microsequences revealed four distinct N‐termini. For one of them, ICL1, N‐terminal and internal peptide sequences were used for PCR amplification and cloning of a DNA fragment containing a complete centrin coding sequence. The deduced amino acid sequence presents about 50% identity with those of centrins from other species. Further molecular analysis allowed us to identify two additional closely related, co‐expressed ICL1 genes, providing the first example of a centrin multigenic family in a microorganism.
C23 Interacts with B23, A Putative Nucleolar‐Localization‐Signal‐Binding Protein
Authors: Li Yi‐Ping , Busch Rose K. , Valdez Benigno C. , Busch Harris
Published: August 2004

The human protein C23 (nucleolin) is a major nucleolar protein. Its interactions with other proteins were studied with the two‐hybrid system which identified nucleolar protein B23 (nucleophosmin) as being associated with C23. Both proteins were co‐immunoprecipitated from HeLa cell nuclear extract by either monoclonal anti‐C23 or monoclonal anti‐B23. Binding studies utilizing deletion mutants indicated that the binding of C23 and B23 involves specific motifs. In addition to an approximately 46‐amino‐acid‐binding domain in B23 (amino acids 194–239), amino acids 540–628 of C23 were required for binding; this region of C23 is required for the nucleolar localization. In addition, nucleolar protein p120 was also found to be co‐immunoprecipitated with B23. A fragment of p120 containing a functional nucleolar localization signal bound to the truncated binding domain of B23, as did C23. These results suggest that the interaction of C23 and B23 may represent a nucleolar‐targeting mechanism in which B23 acts as a nucleolar‐localization signal‐binding protein.
Involvement of Cyclophilin D in the Activation of A mitochondrial Pore by Ca2+ and Oxidant Stress
Authors: Tanveer Ahmed , Virji Sukaina , Andreeva Larisa , Totty Nicholas F. , Hsuan J. Justin , Ward John M , Crompton Martin
Published: August 2004

Heart and liver mitochondria contain a structure that is able to form a large non‐selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclo‐sporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP Photolabelling of a 21‐kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl‐prolyl cis‐trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21‐kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane‐space marker, adenylate kinase. Photolabelling of the 18‐kDa PPIase was unaffected by Ca2+ or ADP. The 21‐kDa PPIase was digested with endoproteinase Asp‐N and 11 of the peptides were N‐terminally sequenced. The sequences were most similar to those of human cyclophilin‐D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.
Cathepsin L is an Intracellular and Extracellular Protease in Paramecium Tetraurelia
Authors: Völkel Helge , Kurz Ursula , Under Jürgen , Klumpp Susanne , Gnau Volker , Jung Günther , Schultz Joachim E.
Published: August 2004

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin‐L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. AH other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 μM.
Malonate Decarboxylase of Klebsiella pneumoniae Catalyses the Turnover of Acetyl and Malonyl Thioester Residues on a Coenzyme‐A‐Like Prosthetic Group
Authors: Schmid Markus , Berg Michael , Hilbi Hubert , Dimroth Peter
Published: August 2004

During aerobic growth of Klebsiella pneumoniae on malonate, a soluble malonate decarboxylase is induced. Malonate decarboxylation consumes a proton (not H2O) and forms acetate and CO2 (not HCO−3) as products. The enzyme was purified 56‐fold to apparent homogeneity. It has a native molecular mass of 142 kDa and consists of four subunits α, β, γ and δ with molecular masses of 65, 34, 30, and 12 kDa, respectively. Two different forms of the enzyme were recognised: a catalytically inactive SH‐enzyme and the catalytically active acetyl‐S‐enzyme which is formed by post‐translational acetylation of the SH‐enzyme with ATP, acetate and a specific ligase. The acetyl‐S‐enzyme was converted into the SH‐enzyme by incubation with hydroxylamine or dithioerythritol. Chemical reacylation of the SH‐enzyme, which restores catalytic activity, was achieved with acetic anhydride or more efficiently with malonyl‐CoA. This acylation of the SH group was prevented after incubation with various thiol‐specific reagents. After incubation of the SH‐enzyme with iodo[1‐14C]acetate, the δ subunit became specifically labelled. This subunit was also labelled after incubation of the acetyl‐S‐enzyme with [2‐14C]malonate. The radioactivity was completely liberated from the protein upon malonate addition. These results indicate that the δ subunit is the acyl‐carrier protein of the complex and that malonate decarboxylation proceeds in two steps: the acetyl residue on the ACP is first replaced by a malonyl residue which subsequently undergoes decarboxylation thereby regenerating the acetyl‐S‐ACP. The binding site for the acyl residues on the acyl carrier protein was shown to be 2′‐(5″‐phosphoribosyl)‐3′‐dephospho‐CoA after alkaline cleavage of this prosthetic group from the enzyme and chromatographic as well as mass spectroscopic analyses.
Characterization of Lipopolysaccharides of Polymyxin‐Resistant and Polymyxin‐Sensitive Klebsiella pneumoniae O3
Authors: Helander Ilkka M. , Kato Yutaka , Kilpeläinen Ilkka , Kostiainen Risto , Lindner Buko , Nummila Kim , Sugiyama Tsuyoshi , Yokochi Takashi
Published: August 2004

Lipopolysaccharides isolated from the polymyxin‐resistant Klebsiella pneumoniae 03 mutant OM‐5 and its polymyxin‐sensitive parent LEN‐1 were analyzed for chemical composition, and their lipid A portions were structurally characterized. The lipopolysaccharide of OM‐5 contained approximately five times more 4‐amino‐4‐deoxy‐l‐arabinopyranose than that of LEN‐1. Other saccharide and phosphate components exhibited no significant differences. Structural characterization, including analyses by phosphorus magnetic resonance spectroscopy and by fast atom bombardment mass spectrometry, revealed a novel type of lipid A. In the OM‐5 lipopolysaccharide, both phosphates of lipid A were almost totally present as phosphodiesters with 4‐amino‐4‐deoxy‐l‐arabinopyranose. In the sensitive‐type LEN‐1 lipid A, the extent of this substitution was much lower, especially in the glycosidically linked phosphate. Phosphate in these K. pneumoniae lipopolysaccharides was almost exclusively found in lipid A. These results show that cationic substituents of phosphates of lipid A play a decisive role in determining polymyxin reactivity. OM‐5 was also found to contain a large proportion of heptaacyl lipid A, which represented only a small fraction of lipid A in LEN‐1.
A (G+C)‐Rich Motif in the Aldolase C Promoter Functions as a Constitutive Transcriptional Enhancer Element
Authors: Cibelli Giuseppe , Schoch Susanne , Pajunk Henrike , Brand Ingeborg A. , Thiel Gerald
Published: August 2004

The enzyme fructose‐l,6‐bisphosphate aldolase consists of three isozymes that are expressed in a tissue‐specific manner. Using antibodies against aldolase B and C., it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)‐rich region of the aldolase C promoter might function as a neuron‐specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143–151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)‐rich sequence, a TATA box, and the rabbit β‐globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the β‐globin mRNA. It is shown that the (G+C)‐rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron‐specific gene expression, directed transcription only in neuronal cells. In gel‐retardation assays, two major DNA‐protein complexes were detected with the (G+C)‐rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA‐binding proteins. These DNA‐protein interactions could be impaired by a DNA probe that contained an Sp1‐binding site, indicating that Sp1 or an Sp1‐related factor binds to the aldolase C promoter (G+C)‐rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr‐l, also known to recognize a (G+C)‐rich consensus site, did not, however, bind to the (G+C)‐rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)‐rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1‐related transcription factors.
Occurrence and Structural Analysis of Highly Sulfated Multiantennary N‐linked Glycan Chains Derived from a Fertilization‐Associated Carbohydrate‐Rich Glycoprotein in Unfertilized Eggs of Tribolodon hakonensis
Authors: Taguchi Tomohiko , Iwasaki Mariko , Muto Yutaka , Kitajima Ken , Inoue Sadako , Khoo Kay‐Hooi , Morris Howard R. , Dell Anne , Inoue Yasuo
Published: August 2004

This study represents the first detailed investigation of the nature of highly sulfated (keratan‐sulfate‐like) complex‐type asparagine‐linked glycans having a tetraantennary core structure and shows the effectiveness of fast‐atom‐bombardment mass spectrometric (FAB‐MS) methods incorporating derivatization and mild methanolysis for analyzing such complex types of sulfated glycans.

The structure of the N‐glycan chains was unambiguously established by a combination of compositional analysis, methylation analysis, mild methanolysis for desulfation, hydrazinolysis/nitrous acid deamination, enzymatic (endo‐β‐galactosidase and peptide: N‐glycosidase F) digestions, and instrumental analyses (1H‐NMR spectroscopy and FAB‐MS) which revealed the novel repeating sulfated carbohydrate sequences, ±Galβ1 → 4Galβ1[→ 4(HSO3→ 6)GlcNAcβ1 → 3(±Galβ1 → 4)Galβ1]n→ (see Structure I; p+q+r+s ≈ 14). This sequence is unique in: (a) the skeletal structure is similar to that of keratan sulfate but is completely devoid of 6‐O‐sulfated Gal residues and (b) the presence of branched Gal residues in the sequence → 4GlcNAcβ1 → 3(Galβ1 → 4)Galβ1→.
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Localization and Regulation of the Putative Membrane Fatty‐Acid Transporter (FAT) in the Small Intestine
Authors: Poirier Hélène , Degrace Pascal , Niot Isabelle , Bernard André , Besnard Philippe
Published: August 2004

The expression of the putative membrane fatty‐acid transporter (FAT) was investigated in the small intestine. The FAT mRNA level was higher in the jejunum than in the duodenum and was lower in the ileum, as observed for cytosolic fatty‐acid‐binding proteins (FABP) expressed in this tissue. No FAT transcript was found in the stomach or colon. FAT mRNA was constitutively expressed in the epithelial cells located in the upper two thirds of villi, while it was undectectable in the crypt cells and submucosal cells. In jejunal mucosa, immunochemical studies showed that FAT protein was limited to the brush border of enterocytes. No fluorescence was found in the goblet cells. To determine whether FAT responded to changes in fat intake, as reported for FABP, the effect of two high‐fat diets, which essentially contained either medium‐chain fatty acids or long‐chain fatty acids (sunflower‐oil diet), was investigated. The sunflower‐oil diet greatly increased FAT mRNA abundance throughout the small intestine. In contrast, a weak effect of medium‐chain fatty acids was observed only in the jejunum. As found for FABP expression, treatment with the hypolipidemic drug bezafibrate affected FAT expression. These data demonstrate that FAT and FABP are co‐expressed in enterocytes, as has been shown in adipocytes, myocytes and mammary cells. The data suggest that these membrane and cytosolic proteins might have complementary functions during dietary‐fat absorption.
The Structure of the Aeromonas proteolytica Aminopeptidase Complexed with a Hydroxamate Inhibitor
Authors: Chevrier Bernard , D'orchymont Hugues , Schalk Céline , Tarnus Céline , Moras Dino
Published: August 2004

The structure of the complex of Aeromonas proteolytica aminopeptidase, a two‐zinc exopeptidase, with the inhibitor p‐iodo‐d‐phenylalanine hydroxamate has been determined by X‐ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80–0.23‐nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N‐hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Zn1, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.
Identification of Residues of Rhodobacter capsulatus Ferredoxin I Important for Its Interaction with Nitrogenase
Authors: Naud Isabelle , Meyer Christine , David Laurent , Breton Jacques , Gaillard Jacques , Jouanneau Yves
Published: August 2004

In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site‐specific mutagenesis. A three‐dimensional structure of FdI was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2→Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI‐deleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild‐type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an E′o of‐510mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe‐4S] ferredoxin. Only the [Tyr2] FdI variant showed a significant difference in redox potential (?E′o=‐15 mV). Based on in vitro assays, a [Glu27, Glu28] FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36→His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36] FdI variant also failed to form a cross‐linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild‐type phenotype when expressed in a FdI‐deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.
Chemical Structure of Lipid A Isolated from Comamonas testosteroni Lipopolysaccharide
Authors: Iida Takatoshi , Haishima Yuji , Tanaka Akira , Nishiyama Katsuhiro , Saito Shigeru , Tanamoto Ken‐ichi
Published: August 2004

The chemical structure of lipid A of lipopolysaccharide isolated from Comamonas testosteroni was determined by quantitative analysis, methylation analysis, mass spectrometry and NMR spectroscopy. The lipid A backbone was found to consist of 6‐O‐(2‐deoxy‐2–amino‐β‐d‐glucopyranosyl)‐2‐deoxy‐2‐amino‐α‐d‐glucose which was phosphorylated in positions 1 and 4′. Hydroxyl groups at positions 4 and 6′ were unsubstituted, and position 6′ of the non‐reducing terminal residue was identified as the attachment site of the polysaccharide part. Liquid secondary‐ion/mass spectrometry revealed a pseudomolecular ion at m/z 1572 [M‐H]− as a major diphosphoryl lipid A component carrying six acyl groups. Fatty acid distribution analysis and electrospray ionization/mass spectrometry of the lipid A showed that positions 2, 2′, 3 and 3′ of the sugar backbone were N‐acylated or O ‐acylated by (R)‐3‐hydroxydecanoic acid, and that the hydroxyl groups of the amide‐linked residues attached to positions 2 and 2′ were further O‐ acylated by tetradecanoic and dodecanoic acids, respectively.
The Identification of 11β‐hydroxysteroid Dehydrogenase as Carbonyl Reductase of the Tobacco‐Specific Nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone
Authors: Maser Edmund , Richter Elmar , Friebertshäuser Jutta
Published: August 2004

The tobacco‐specific nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), which is generated by nitrosation of nicotine, requires enzymatic activation by cytochrome‐P450‐mediated α‐carbon hydroxylation to yield particularly powerful carcinogenic alkylating intermediates. Pyridine‐N‐oxidation and carbonyl reduction are detoxification pathways, the latter by providing the functional hydroxy moiety necessary for glucuronosylation and final excretion of NNK. For more than a decade, the enzyme responsible for NNK carbonyl reduction has awaited characterization. In the present study, we demonstrate that the NNK carbonyl reductase is identical to 11β‐hydroxysteroid dehydrogenase (11β‐HSD), the physiological function of which is the oxidoreduction of glucocorticoids. We conclude that the expression of 11 β‐HSD (together with glucuronosyl transferase) may have profound influence on the carcinogenic potency of NNK and that many compounds of endogenous and exogenous origin, which are known to be substrates or inhibitors of 11β‐HSD, may modulate NNK‐induced carcinogenicity by competing for the same enzyme. In light of the known species and tissue differences in the expression of 11β‐HSD isozymes, important aspects of NNK‐induced tumorigenesis, such as metabolic activation versus inactivation or organospecificity, can now be re‐evaluated.
11β‐Hydroxylase Activity in Recombinant Yeast Mitochondria
Authors: Dumas Bruno , Cauet Gilles , Lacour Thierry , Degryse Eric , Laruelle Laurence , Ledoux Catherine , Spagnoli Roberto , Achstetter Tilman
Published: August 2004

In mammals, the final 11β‐hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P‐45011β, together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11β‐hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P‐45011β whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH‐adrenodoxin‐reductase‐like activity. Adrenodoxin and P‐45011β behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P‐45011β. The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH.

The notion that a yeast microsomal NADPH P‐450 oxidoreductase can transfer electrons to mammalian microsomal P‐450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P‐450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.
Characterization of the Functional Gene Encoding Mouse Class III Alcohol Dehydrogenase (Glutathione‐Dependent Formaldehyde Dehydrogenase) and An Unexpressed Processed Pseudogene with An Intact Open Reading Frame
Authors: Foglio Mario H. , Duester Gregg
Published: August 2004

Multiple forms of vertebrate alcohol dehydrogenase (ADH) have been identified, but only one form, class III ADH, has been conserved in all organisms studied. Class III ADH functions in vitro as a glutathione‐dependent formaldehyde dehydrogenase, which suggests that this was the original function that drove the evolution of ADH. Genetic analysis of class III ADH in yeast supports this view, but such studies are lacking in higher eukaryotes. The mouse ADH family has been previously analyzed and it contains three forms of ADH including the class III enzyme. We have initiated a molecular genetic analysis of the mouse class III ADH gene (Adh‐2) by screening a genomic library with a full‐length cDNA. Two overlapping clones contained the complete Adh‐2 gene composed of nine exons in a 12‐kb region, with the placement of introns matching that observed in other mammalian ADH genes. In this screening, we also isolated a clone (ψAdh‐2) that lacks introns and which resembles a processed pseudogene. ψAdh‐2 contained 25 point mutations relative to the previously analyzed Adh‐2 cDNA, but still retained an intact open reading frame. Northern blot analysis using gene‐specific probes provided evidence that ψAdh‐2 does not produce a mRNA in either liver or kidney, whereas Adh‐2 does. The functionality of the two genes was also compared by fusion of their 5′‐flanking regions to a lacZ reporter gene. Reporter gene expression following transfection into mouse F9 embryonal carcinoma cells indicated that only Adh‐2 possesses promoter activity.
Study of the Coordination Chemistry of Prostaglandin G/H Synthase by Resonance Raman Spectroscopy
Authors: Gaspard Sarra , Chottard Geneviève , Mahy Jean‐Pierre , Mansuy Daniel
Published: August 2004

Resonance Raman spectra of prostaglandin G/H synthase (PGHS) in its ferric and ferrous states have been obtained by Soret excitation. In native PGHS, which contained only 0.25 heme/monomeric apoprotein, the ferric heme was in a high‐spin hexacoordinated state. The presence of a vibration at 489 cm‐1 that was responsive to H216O→H218O replacement was taken as evidence for the presence of a H‐bonded H2O molecule as the sixth ligand of the Fe. A study, by CD and resonance Raman spectroscopy, of heme incorporation into the apoprotein showed that, for heme/protein ratios lower than 0.5, the heme was in the same ferric high‐spin hexacoordinated state as in the native enzyme. For heme/protein ratios higher than 0.5, the concomitant formation of two minor species was observed: a low‐spin hexacoordinated species which could be due to the axial coordination of a distal histidine to the Fe trans to its proximal histidine ligand; and a high‐spin pentacoordinated species that corresponded to non‐specific binding of the heme to the apoprotein.

In the reduced state, the heme of PGHS contained a high‐spin pentacoordinated Fe(II) with a histidine as the proximal ligand. However, this species shifted spontaneously towards a low‐spin hexacoordinated Fe(II) species in which the iron was probably coordinated by a distal histidine as the sixth axial ligand. The PGHS Fe(II) · CO derivative displayed an Fe‐CO stretching mode at 529 cm−1, which is in the range observed for peroxidases. Such a high frequency could be due to H‐bonding between the oxygen atom of the CO ligand and the distal histidine, His207. Since this histidine plays an important role, by coordination of Fe(II) or Fe(III) of PGHS and stabilization of the ligands of the Fe, H2O or CO by H‐bonding, it is suggested that this histidine could also play a key role in the cleavage of the O‐O bond of peroxides by peroxidases.
Crystal Structure of Pig Pancreatic α‐amylase Isoenzyme II, in Complex with the Carbohydrate Inhibitor Acarbose
Authors: Gilles Coralie , Astier Jean‐Pierre , Marchis‐Mouren Guy , Cambillau Christian , Payan Françoise
Published: August 2004

Two different crystal forms of pig pancreatic α‐amylase isoenzyme II (PPAII), free and complexed to a carbohydrate inhibitor (acarbose), have been compared together and to previously reported structures of PPAI. A crystal form obtained at 4°C, containing nearly 72% solvent, made it possible to obtain a new complex with acarbose, different from a previous one obtained at 20°C [Qian, M., Buisson, G., Duée, E., Haser, H. & Payan, F. (1994) Biochemistry 33, 6284–6294]. In the present form, six contiguous subsites of the enzyme active site are occupied by the carbohydrate ligand; the structural data indicate that the binding site is capable of holding more than the five glucose units of the scheme proposed through kinetic studies. A monosaccharide ring bridging two protein molecules related by the crystal packing is located on the surface, at a distance of 2.0 nm from the reducing end of the inhibitor ligand; the symmetry‐related glucose ring in the crystal lattice is found 1.5 nm away from the non‐reducing end of the inhibitor ligand.