bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilization of the tetrameric and monomeric forms of pigeon liver malic enzyme on Sepharose beads
Authors: CHANG Gu‐Gang , HUANG Ter‐Mei , CHANG Tsu‐Chung
Published: March 2005

Pigeon liver malic enzyme was chemically attached to Sepharose 4B‐CL beads. The enzyme lost ∼ 50% of its original activity when immobilization was carried out with 5 mg CNBr/ml gel. Immobilization performed at pH 8.0 or pH 4.5 resulted in the formation of matrix‐bound tetramer and monomer, respectively. Matrix‐bound reconstituted tetramer was derived from matrix‐bound monomer by mixing the latter with soluble enzyme at pH 4.5, then raised the pH of the solution to 8.0. The matrix‐bound monomer was demonstrated to be enzymically fully active in terms of specific activity. The pH profile for the enzymic reaction was similar for both soluble and immobilized enzymes. However, the latter had a broader range for the optimum pH (pH 6.8–7.8). The Arrhenius plots for all immobilized enzyme forms were biphasic with inflection at ∼ 27°C. The apparent Michaelis constants for the substrates increased about 2–3‐fold after immobilization. All immobilized enzymes forms, including the matrix‐bound monomer, showed substrate inhibition at high concentrations of L‐malate. Both high‐affinity and low‐affinity binding sites for Mn2+ existed for all immobilized enzyme forms. These results are consistent with an existing asymmetric model, but are not compatible with a sequential model for the enzyme tetramer. The immobilized enzyme was stable for at least four months at 4°C. As compared to soluble enzyme, the immobilized enzyme was less inhibited by (NH4)2SO4 or NaCl. It was also resistant to inactivation with periodateoxidized aminopyridine adenine dinucleotide phosphate, an affinity label for malic enzyme. Incubation of the immobilized enzyme (1.25 μM) with the reagent (5.6 mM) resulted in pseudo‐firstorder inactivation with a rate constant of 0.0108 min−1 that was at least an order of magnitude smaller than that for the soluble enzyme.
Prostaglandin‐E1‐binding sites in rabbit erythrocyte membranes
Authors: DUTTA‐ROY Asim K. , HOQUE Lutful , PATERSON Brian J.
Published: March 2005

Prostaglandin E1 (PGE1) binding sites have been identified on rabbit erythrocyte membranes. The binding of PGE1 to the membranes was found to be highly specific, reversible, and saturable. The high‐affinity binding sites had a dissociation constant (Kd,1) of 5.6 ± 1.2 nM with a binding capacity of 210 ± 51 fmol/mg protein, whereas the low‐affinity binding sites had a dissociation constant (Kd,2) of 22 ± 6.4 μM, and a binding capacity of 321 ± 78 pmol/mg protein. Incubation with PGE1 did not activate adenylate cyclase in the membranes. Preincubation of rabbit erythrocyte membranes with physiological amounts of insulin (1.5 nM) resulted in an increase of PGE1 binding to the membranes from 241 ± 65 to 429 ± 85 fmol/mg protein. The insulin‐induced increase in PGE1 binding was due to an increase in binding sites (both high‐affinity and low‐affinity binding sites) rather than to an increase in the affinity of the binding sites. Treatment of erythrocyte membranes with PGE1 at concentrations (4.0–7.5 nM) which were within the Kd,1 value of the high‐affinity binding sites, resulted in a significant reduction in membrane fluorescence anisotropy (0.27 ± 0.005–0.21 ± 0.003). Use of higher concentrations (>15 nM) of PGE1 reversed the effect of its lower concentration on the membrane anisotropy.
X‐ and Q‐band EPR studies on the two Mn2+‐substituted metal‐binding sites of d‐xylose isomerase
Authors: BOGUMIL Ralf , KAPPL Reinhard , HÜTTERMANN Jürgen , SUDFELDT Christoph , WITZEL Herbert
Published: March 2005

The two metal‐binding sites (A and B)/subunit of the homotetrameric d‐xylose isomerase (Xyl isomerase) from Streptomyces rubiginosus have been studied with Mn2+‐EPR spectroscopy at X‐band and Q‐band frequencies and with electronic spectroscopy. Displacement studies in the visible absorbance range showed that Mn2+ have a higher affinity for the B site. With the low‐affinity A site unoccupied, the coordination sphere of Mn2+ in the B site is quite distorted giving rise to a highly anisotropic X‐band EPR spectrum. Simulation of the Q‐band spectrum reveals a zero field splitting (zfs) D of about 45–48 mT and a rhombicity parameter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn2+ induces a significant shift towards a higher symmetry in the coordination sphere of the B site resulting in similar zfs parameters for both binding sites. The change in A‐site environment caused by B‐site occupation was analysed in mixed Xyl isomerase derivatives, in which the B site is loaded with Co2+, Cd2+ or Pb2+ and the A site with Mn2+. In the Co2+/Mn2+ Xyl isomerase the Mn2+ has a relatively symmetric ligand environment with small zfs parameters (D= 12 mT, E/D < 0.15). Substituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increase in the zfs parameters of Mn2+ in the A site. The distortions are directly linked to the ionic radii of the ions bound to the B site and may be mediated by the carboxylate group of Glu216 that bridges the metal‐binding sites. The EPR spectra also reflect the catalytic activity of the mixed metal samples. With the larger Cd2+ or Pb2+ in the B site, which are strongly influencing the stereochemistry of the A site, the catalytic activity is lost, whereas Co2+ and Mn2+ render the enzyme in an active state, so that the mutual influence on catalysis depends on the complex geometry of both metal‐binding sites.
Lactotransferrin binding to its platelet receptor inhibits platelet aggregation
Authors: LEVEUGLE Béatrice , MAZURIER Joël , LEGRAND Dominique , MAZURIER Claudine , MONTREUIL Jean , SPIK Geneviève
Published: March 2005

A fluorescent lactotransferrin probe was prepared by coupling 5‐({[2‐(carbhydrazino)methyl]‐thio}acetyl)amino fluorescein to aldehyde groups that were produced by a mild periodic‐acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor‐binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. The fluorescent probe allowed us to characterize, by flow cytometry, the binding of lactotransferrin to non‐activated human platelets. The putative lactotransferrin platelet receptor was purified and its immunological and physico‐chemical properties were found to be very similar to those of the receptor previously isolated from activated human lymphocytes. Lactotransferrin inhibits ADP‐induced platelet aggregation at concentrations down to 5 nM, which can be reached in the plasma after leukocyte degranulation. Inhibition of platelet aggregation was also observed with the N‐terminal fragment of lactotransferrin (residues 3–281; 50% inhibition = 2 μM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20–37; 50% inhibition = 20 μM) corresponding to one of the two external loops (residues 28–34 and 39–42) where we recently located the receptor‐binding site. The activity (50% inhibition = 500 μM) of the tetrapeptide KRDS (residues 39–42), which has already been described, was at least 25‐times and 16000‐times lower than the activity of the octodecapeptide and of the lactotransferrin molecules, respectively. Finally, the inhibition was demonstrated to be mediated by a mechanism which requires the binding of lactotransferrin to its putative receptor and not to platelet glycoprotein IIb‐IIIa.
Gastric chief cell‐specific transcription of the pepsinogen A gene
Authors: MEIJERINK Peter H. S. , BEBELMAN Jan‐Paul , OLDENBURG Anja M. , DEFIZE Jacques , PLANTA Rudi J. , ERIKSSON Aldur W. , PALS Gerard , MAGER Willem H.
Published: March 2005

The molecular mechanisms underlying the regulation of pepsinogen A (PGA) gene expression in mammalian cells are poorly understood. In this paper we describe the structural and functional analysis of the pepsinogen A gene promoter in the pig. By genomic Southern analyses we demonstrate that, in contrast with human PGA genes which are amplified and organized in haplotypes, only a single PGA gene is present per haploid porcine genome. With the aim of identifying promoter elements mediating the gastric mucosa cell‐specific transcription of the PGA gene in pig, we isolated a PGA gene from a porcine genomic library. The nucleotide sequence of the first exon and 1.7 kb of the upstream DNA region were determined and compared with the corresponding regions of the human PGA gene encoding isozymogen Pg5. In order to study the promoter activity of the PGA gene a functional assay was developed: we succeeded in obtaining primary monolayer cultures of porcine gastric mucosal chief cells, suitable for transfection. Fragments of 5′‐flanking and noncoding first exon sequences of the porcine and human PGA genes were linked to the chloramphenicol acetyltransferase (CAT) gene. The transcriptional activity of these hybrid genes was assessed in transient expression assays upon transfection (lipofection) of gastric and nongastri cells. Whereas PGA 5′‐flanking sequences showed no promoter activity in nongastric cell types, the DNA region from −205 to + 21 was found to be sufficient to direct expression of the porcine PGA constructs in a cell‐specific manner. Further deletion analysis of the proximal promoter fragment identified several regions (−205 to −167, −127 to −67 and +2 to +21) acting synergistically in the transcriptional regulation of the PGA gene. In contrast, all human PGA‐CAT constructs used failed to show promoter activity in porcine gastric chief cells, indicating species‐specific control of PGA gene expression. In addition, the transcriptional activity of the porcine PGA promoter in chief cells from pig was completely abolished by in vitro CpG methylation. Footprint analyses of the proximal promoter fragment using nuclear extracts from either porcine gastric mucosal chief cells or liver revealed some notable differences between both extracts, which might reflect the interaction with (a) cell‐specific factor(s).
Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum
Authors: SONNTAG Kirsten , EGGELING Lothar , DE GRAAF Albert A. , SAHM Hermann
Published: March 2005

The Gram‐positive Corynebacterium glutamicum has the potential to synthesize l‐lysine via a split pathway, where amino‐ketopimelate is converted to the ultimate lysine precursor diaminopimelate either by reactions involving succinylated intermediates, or by one single reaction catalysed by D‐diaminopimelate dehydrogenase. To quantify the flux distribution via both pathways, 13C‐enriched glucose was used and specific enrichments in lysine and in pyruvate‐derived metabolites were determined by 13C‐and 1H‐NMR spectroscopy. Using a system of linear equations, the contribution of the D‐diaminopimelate dehydrogenase pathway was determined to be about 30% for the total lysine synthesized. This was irrespective of whether lysine‐accumulting mutants or the wild‐type strain were analysed. However, when the distribution was determined at various cultivation times, the flux partitioning over the dehydrogenase pathway in a producing strain decreased from 72% at the beginning to 0% at the end of lysine accumulation. When ammonium sulphate was replaced by the organic nitrogen source glutamate, the ammonium‐dependent D‐diaminopimelate dehydrogenase pathway did not contribute to total lysine synthesis at all. Additional experiments with varying initial ammonium concentrations showed that in Corynebacterium glutamicum the flux distribution over the two pathways of lysine synthesis is governed by the ammonium availability. This is thus an example where an anabolic pathway is directly influenced by an extrcellular medium component, probably via the kinetic charcteristics of D‐diaminopimelate dehydrogenase.
Predicting the topology of eukaryotic membrane proteins
Authors: SIPOS Laszlo , VON HEIJNE Gunnar
Published: March 2005

We show that the so‐called ‘positive inside’ rule, i.e. the observation that positively charged amino acids tend to be more prevalent in cytoplasmic than in extra‐cytoplasmic segments in transmembrane proteins [von Heijne, G. (1986) EMBO J. 5, 3021–3027], seems to hold for all polar segments in multi‐spanning eukaryotic membrane proteins irrespective of their position in the sequence and hence can be used in conjunction with hydrophobicity analysis to predict their transmembrane topology. Further, as suggested by others, we confirm that the net charge difference across the first transmembrane segment correlates well with its orientation [Hartmann, E., Rapoport, T. A. and Lodish, H. F. (1989) Proc. Natl Acad. Sci. USA 86, 5786–5790], and that the overall amino‐acid composition of long polar segments can also be used to prodict their cytoplasmic or extra‐cytoplasmic location [Nakashima, H. and Nishikawa, K. (1992) FEBS Lett. 303, 141–146]. We present an approach to the topology prediction problem for eukaryotic membrane proteins based on a combination of these methods.
A novel very small subunit of a selenium containing [NiFe] hydrogenease of Methanococcus voltae is postranslationally processed by cleavage at a defined position
Authors: SORGENFREI Oliver , LINDER Dietmar , KARAS Michael , KLEIN Albrecht
Published: March 2005

A coenzyme‐F420 non‐reducing [NiFe] hydrogenase was isolated from Methanococcus voltae. It consist of three subunits. They are the products of the prevously identified genes vhuA, vhuG and vhuU. The vhuU gene product is of only 25 amino acids. This novel very small hydrogenase subunit contains selenocysteine within a conserved amino‐acid sequence previously shown to be involved in Ni coordination. The subunit is shorter than the predicted primary gene product and is therefore apparently post‐translationally processed.
Structural analysis of periplasmic carbonic anhydrase 1 of Chlamydomonas reinhardtii
Authors: ISHIDA Sarahmi , MUTO Shoshi , MIYACHI Shigetoh
Published: March 2005

Periplasmic carbonic anhydrase 1 of the unicellular green alga Chlamydomonas reinhardtii is a heterotetrameric glycoprotein consisting of two large subunits and two small subunits [Kamo, K., Shimogawara, K., Fukuzawa, H., Muto, S. & Miyachi, S. (1990) Eur. J. Biochem. 192, 557–562]. The cDNA sequence showed that the two subunits are cotranslated as a 377‐amino‐acid precursor polypeptide (41.6 kDa) consisting of an N‐terminal 20‐amino‐acid signal peptide, a large subunit (35.6 kDa) and a small subunit (4.1 kDa) [Fukuzawa, H., Fujiwara, S., Yamamoto, Y., Dionisio Sese, M. L. & Miyachi, S. (1990) Proc. Natl Acad. Sci. USA 87, 4383–4387]. In the present study, amino‐acid‐sequence analysis of the carbonic anhydrase 1 was carried out to determine the sites of disulfide bonds and N‐glycosylation and the C‐terminal amino acid of the large subunit. Disulfide bonds were detected between Cys21 and Cys21, and Cys61 and Cys264, Cys194 and Cys198, and Cys296 and Cys351 in the amino acid sequence deduced from the cDNA. Cys21 and Cys21 link two large subunits and Cys296 and Cys351 link a large subunit to a small subunit. Thus, the holoenzyme is constructed of two disulfied‐bound large subunits, each of which joins to a small subunit by a disulfide bond. The C‐terminal amino acid residue of the large subunit was determined as Ala305. This indicates that the peptide consisting of 35 amino acid residues between the large and the small subunit is deleted from the precursor during maturation, since the N‐terminus of the small subunit is Ala341. Three potential N‐glycosylation sites in the large subunit were all glycosylated.
Molecular cloning and nucleotide sequence of cDNA encoding rat kidney long‐chain l‐2‐hydroxy acid oxidase
Authors: BELMOUDEN Ahmed , LÊ K. H. Diêp , LEDERER Florence , GARCHON Henri‐Jean
Published: March 2005

Long‐chain l‐α‐hydroxy acid oxidase from rat kidney is a member of the family of FMN‐dependent α‐hydroxy‐acid‐oxidizing enzymes. With the knowledge of the recently determined amino acid sequence, the cDNA encoding the enzyme has now been cloned using the polymerase chain reaction. The 1648‐bp cDNA contains an open reading frame coding for the 352 residues of the previously determined sequence, preceded by a methionine codon. In addition, several clones were found to present a nine‐base insertion, predicting the existence of an isoform with a tripeptide VRK inserted between residues 188 and 189 of the mature protein. The presence of about 10% of this isoform in the oxidase purified from rat kidney was indeed identified by amino acid sequencing. A recombinant active enzyme was obtained as a protein fused to glutathione S‐transferase using the bacterial expression plasmid pGEX‐3X. Physico‐chemical characterization indicated, for the fused enzyme, properties similar to those of the rat kidney protein. When the chimaera was submitted to factor Xa, proteolysis at the engineered cleavage point was poor. Separation of hydroxy acid oxidase from glutathione' S‐transferase could not be achieved with trypsin either. With both proteases, the initial cleavage point appeared to be in a peptide loop internal to the hydroxy acid oxidase sequence, close to or in the tripeptide insertion locus and not at the engineered factor‐Xa‐cleavage point. Comparative tryptic proteolysis of the rat kidney enzyme yielded a form cleaved in the same loop.
Functional expression of d‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima in Escherichia coli
Authors: TOMSCHY Andrea , GLOCKSHUBER Rudi , JAENICKE Rainer
Published: March 2005

The gene coding for d‐glyceraldehyde‐3‐phosphate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima has been cloned and functionally expressed in Escherichia coli. Some 90% of the coding gene was amplified by the polymerase chain reaction. The amplified gene segment was in full agreement with the previously determined amino acid sequence [Schultes, V., Deutzmann, R., Jaenicke, R. (1990) Eur. J. Biochem. 192, 25–31] and was completed by the insertion of synthetic linkers using site‐directed mutagenesis. The resulting semisynthetic gene was expressed in high yield in the cytoplasm of E. coli and the recombinant enzyme was purified to homogeneity. It was shown to be identical with the enzyme isolated directly from T. maritima in all enzymatic and physicochemical properties investigated. The enzyme is allosterically inhibited by both d‐and l‐glyceraldehyde 3‐phosphate at concentrations above 1 mM, and by arsenate at concentrations above 10 mM. The expressed protein restores the natural E. coli phenotype in a gap strain, thus providing evidence that the hyperthermophilic protein can fold and associate to its native, functional state in its mesophilic host.
Mass determination of 15‐hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)‐12‐hydroxy‐5,8,10‐heptadecatrienoic acid as substrate
Authors: HÖHL Wolfgang , STAHL Bernd , MUNDKOWSKI Ralf , HOFMANN Ute , MEESE Claus O. , KUHLMANN Ulrich , SCHLEGEL Werner
Published: March 2005

NAD+‐dependent 15‐hydroxyprostaglandin dehydrogenase catalyzes the first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have examined enzyme kinetics of the purified enzyme with (5Z,8E,10E,12S)‐12‐hydroxy‐5,8,10‐ heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtration on FPLC. We could estimate a molecular mass of 28 ± 1 kDa, indicating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix‐assisted laser desorption/ionization mass spectrometry to 28740 ± 30 Da.

(5Z,8E,10E)‐12‐oxo‐5,8,10‐heptadecatrienoic acid (oxo‐HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 μM, Vi= 12.78 mU/μg] were in agreement with those determined for NADH formation (Km= 7.65 μM, Vi= 11.79 mU/μg). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 (PGE2). As the product oxo‐HT is a potent inhibitor of platelet aggregation, dehydrogenation of HHT might represent a biological activation step.
Molecular characterization of Limulus polyphemus C‐reactive protein
Authors: TENNENT Glenys A. , BUTLER P. Jo G. , HUTTON Thérèse , WOOLFITT Adrian R. , HARVEY David J. , RADEMACHER Thomas W. , PEPYS Mark B.
Published: March 2005

The molecular mass of whole native C‐reactive protein (CRP) from Limulus polyphemus, the horseshoe crab, was precisely determined for the first time, by analytical ultracentrifugation after measurement of its absorption coefficient, A1%1cm, at 280 nm (15.49) and its partial specific volume (0.72 ± 0.003 ml/g). The apparent weight‐average molecular mass, in the range over which it was independent of the concentration, was 300 kDa. Limulus CRP, isolated from an individual animal, was also analyzed by electrospray mass spectrometry after dissociation into its subunits, and nine components were detected in the mass range 24935–25617 Da. Four of these components corresponded precisely to one of the previously reported protein sequences of Limulus CRP subunits which is glycosylated by a single oligosaccharide chain of composition, Man2GlcNAc2, Man3GlcNAc2, Man4GlcNAc2 or Man5GlcNAc2, the structures of which are elucidated in the accompanying paper [Amatayakul‐Chantler, S., Dwek, R. A., Tennent, G. A., Pepys, M. B. & Rademacher, T. W. Molecular characterization of Limulus polyphemus C‐reactive protein. II. Asparaginelinked oligosaccharides (1993) Eur. J. Biochem. 214, 99–110]. All the remaining components, except one minor component, corresponded to the analogous glycoforms of peptides of molecular mass 24281 ± 2Da and 24523 ± 1Da, neither of which have previously been characterized. These results are consistent with the presence of 12 subunits in the Limulus CRP molecule, in contrast to the number of subunits in vertebrate members of the CRP family. The CRP family members in vertebrates are known as pentraxins because of the symmetrical pentameric arrangement of their subunits. The concentration of Limulus CRP in hemolymph from 20 individual animals, measured precisely by specific electroimmunoassay, covered the wide range 0.275–6.64 mg/ml, mean (SD)=1.83 (2.06) mg/ml. It remains to be determined whether this protein is an acute‐phase reactant, like its human and some other mammalian homologues.
The glucose‐6‐phosphate‐isomerase reaction is essential for normal glucose repression in Saccharomyces cerevisiae
Authors: SIERKSTRA Laurens N. , SILLJÉ Herman H. W. , VERBAKEL John M. A. , VERRIPS C. Theo
Published: March 2005

Wild‐type Saccharomyces cerevisiae and a strain carrying a deletion in the glucose‐6‐phosphate‐isomerase gene (pgi1) were grown in carbon‐limited continuous cultures on a mixture of fructose and galactose. Pulses of glucose, fructose and galactose were given to these cultures to investigate whether the pgi1 strain was capable of normal glucose repression. Glucose and galactose pulses inhibited fructose consumption and thus glycolysis in the pgi1 strain by a combination of competition between glucose and fructose at the uptake and/or phosphorylation level and inhibition of fructose uptake and/or phosphorylation by glucose 6‐phosphate. Fructose pulses administered to the pgi1 strain transiently decreased the glycolytic flux downstream of fructose‐1,6‐bisphosphate. Transcriptional induction of the PDCl gene (encoding pyruvate decarboxylase) was observed after glucose or galactose pulses were applied to the pgi1 strain, demonstrating that metabolism of these sugars beyond glucose 6‐phosphate is dispensable for PDCl induction. Fructose also induced PDCl transcription, indicating that intracellular sugars could act as trigger for PDCl induction or, alternatively, that two inductors are present. In contrast to the wild‐type transcriptional inhibition of the glucose‐repressible genes, HXK1 and GAL10 (encoding hexokinase isoenzyme 1 and uridine diphosphoglucose 4‐epimerase, respectively) did not occur upon addition of glucose or fructose to the pgi1 mutant. Transcriptional repression was observed after application of the fructose pulse when the yeast had resumed metabolism of fructose. These results demonstrate that the initial signal for catabolite repression is not generated by high sugar concentrations or high concentrations of intermediates; moreover a simple role for the hexokinases can also be excluded. The absence of an increased glycolytic flux in the pgi1 mutant after administration of the sugar pulses while the concentrations of sugar and glycolytic intermediates were high, suggests that the initial signal for glucose repression could be linked to an increased glycolytic flux. The occurrence of PDCl induction in the pgi1 strain while GAL101HXK1 repression is absent, demonstrates that the initial signals for catabolite induction and catabolite repression are different.
Interactions between glucose metabolism and oxidative phosphorylations on respiratory‐competent Saccharomyces cerevisiae cells
Authors: BEAUVOIT Bertrand , RIGOULET Michel , BUNOUST Odile , RAFFARD Gérard , CANIONI Paul , GUÉRIN Bernard
Published: March 2005

The purpose of this work was to analyze the interactions between oxidative phosphorylations and glucose metabolism on yeast cells aerobically grown on lactate as carbon source and incubated in a resting cell medium. On such respiratory‐competent yeast cells, four different metabolic steady states have particularly been studied: (a) glucose feeding under anaerobiosis, (b) ethanol supply under aerobiosis, (c) glucose supply under aerobiosis and (d) glucose plus ethanol under aerobiosis. For each condition, we measured: (a) the cellular ATP/ADP ratio and NADH content sustained under these conditions, (b) the glucose consumption rate (glucose conditions) and the respiratory rate (aerobic conditions).

Under aerobic conditions, when ethanol is used as substrate, the ATP/ADP ratio and NADH level are very high as compared with glucose feeding. However, the rate of oxygen consumption is similar under both conditions. The main observation is a large increase in the respiratory rate when both glucose and ethanol are added. This increase corresponds to an ATP/ADP ratio and a NADH level lower than those observed with ethanol but higher than those with glucose. Therefore the response of the respiratory rate to the ATP/ADP ratio depends on the redox potential. We studied the way in which the ATP‐consuming activity was increased under glucose + ethanol conditions. By NMR experiments, it appears that neither the futile cycle at the level of the phosphofructo‐1‐kinase/fructo‐1,6‐bisphosphatase couple nor the synthesis of carbohydrate stores could account for the increase in oxidative phosphorylation. However, it is shown that, in the presence of glucose + ethanol, ATP consumption is strongly stimulated. It is hypothesized that this consumption is essentially due to the combination of the well‐known plasma membrane proton‐ATPase activation by glucose and the high phosphate potential due to oxidative ethanol metabolism. While it is well documented that oxidative phosphorylations inhibit the glycolytic flux, i.e. the Pasteur effect, we clearly show in this work that the glycolytic pathway limits the ability of mitochondria to maintain a cellular phosphate potential.
Characterization of the Actinobacillus pleuropneumoniae serotype K11:01 capsular antigen
Authors: BEYNON Linda M. , RICHARDS James C. , PERRY Malcolm B.
Published: March 2005

The capsular antigen of Actinobacillus pleuropneumoniae serotype 11 was characterized by one‐dimensional and two‐dimensional high‐field nuclear magnetic resonance methods and chemical analyses, as a teichoic‐acid‐type polymer composed of a repeating unit with the structure

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Molecular cloning, sequence analysis and expression of the gene for catalase‐peroxidase (cpeA) from the photosynthetic bacterium Rhodobacter capsulatus B10
Authors: FORKL Hubert , VANDEKERCKHOVE Joel , DREWS Gerhart , TADROS Monier Habib
Published: March 2005

The gene encoding catalase‐peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7‐kb SacI–HindIII fragment, containing the catalase‐peroxidase gene (cpeA) and its flanking regions were determined. A 1728‐bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed. A Shine‐Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase‐peroxidase of R. capsulatus B10.

The amino acid sequence of R. capsulatus catalase‐peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase‐peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase‐peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.
Structural analysis of O4‐reactive polysaccharides from recombinant Escherichia coli
Authors: KOGAN Grigorij , HARAGUCHI Gayle , HULL Sheila I. , HULL Richard A. , SHASHKOV Alexander S. , JANN Barbara , JANN Klaus
Published: March 2005

In previous studies it had been shown that lipopolysaccharide from O4‐specific recombinant Escherichia coli, had serological reactivities and a chemical composition that differed from wild‐type O4 LPS [Haraguchi, G. E., Zähringer, U., Jann, B., Jann, K., Hull, R. A. & Hull, S. I. (1991) Microb. Pathog. 10, 351–361]. Here we present the structural elucidation of the O‐specific moieties from lipopolysaccharides of some of the recombinant strains obtained in previous studies. Compositional analysis, methylation, chemical reactions and NMR spectroscopy showed that, during genetic manipulations (recombination, cosmid cloning, plasmid subcloning), a gradual structural change in the O‐specific polysaccharides was observed in the recombinant strains. These changes comprised of an alteration in the position of glucose (side chain) substitution, a change in the anomeric configuration of the main‐chain N‐acetylglucosamine and an exchange of α‐l‐rhamnopyranose for β‐d‐galactofuranose. The relevance of these results for lipopolysaccharide cloning and lipopolysaccharide biosynthesis are discussed.
Chemoattractants for neutrophils in lipopolysaccharide‐induced inflammatory exudate from rats are not interleukin‐8 counterparts but gro‐gene‐product/melanoma‐growth‐stimulating‐activity‐related factors
Authors: WATANABE Kazuyoshi , IIDA Megumi , TAKAISHI Kiyosumi , SUZUKI Tadaaki , HAMADA Yoko , IIZUKA Yoshio , TSURUFUJI Susumu
Published: March 2005

Potent chemotactic activity for neutrophils was detected in rat inflammatory exudate induced by a subcutaneous injection of lipopolysaccharide in a carboxymethyl‐cellulose suspension. We purified and characterized chemoattractants from the exudate by the following procedures: carboxymethyl Sephadex C‐25 ion‐exchange chromatography; G3000SW gel‐filtration chromatography; preparative reverse‐phase high‐pressure liquid chromatography; rechromatography on reverse‐phase HPLC. Two chemotactic factors were purified and their N‐terminal amino acid sequences were determined. One factor was a protein in which the first 20 N‐terminal amino acids were identical to those of rat cytokine‐induced neutrophil chemoattractant (CINC), a counterpart of human gro/melanoma growthstimulating activity (MGSA). The other factor was highly similar to mouse macrophage inflammatory protein 2 (MIP‐2). Mouse MIP‐2, a chemotactic factor for neutrophils, is a member of the interleukin‐8 family; however the protein we purified had higher similarity to human gro/MGSA than to human interleukin‐8. These results indicate that, in rats, chemotactic factors for neutrophils induced by lipopólysaccharide stimulation are not counterparts of interleukin‐8, but are gro/CINC‐related peptides.
Zn‐exchange and Mössbauer studies on the [Fe‐Fe] derivatives of the purple acid Fe(III)‐Zn(II)‐phosphatase from kidney beans
Authors: SUERBAUM Hildegard , KÖRNER Michael , WITZEL Herbert , ALTHAUS Erika , MOSEL Bernd‐Dieter , MÜLLER‐WARMUTH Werner
Published: March 2005

In order to perform Mössbauer studies, Zn(II) in the Fe(III)‐Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)‐57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)‐57Fe(III) species with a 30‐times lower activity. Incubation of the metal‐free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)‐57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)‐57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)‐Fe(II)/Fe(III)‐Fe(III) species, but not the mixed valent Fe(III)‐Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III).

The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with δ of 0.51 mm/s and 0.53 mm/s and E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a μ‐oxo bridge. The mixed‐valent Fe(III)‐Fe(II) species shows two quadrupole doublet with δ values of 0.55 mm/s and 1.14 mm/s and E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half‐integer spin system with antiferromagnetic coupling. The Fe(II)‐Fe(II) system exhibits two quadrupole doublets with δ values of 1.18 mm/s and 1.22 mm/s and with E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)‐binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)‐Fe(II) system. Interaction with O2 is discussed.
Characterization of a transcriptional control element involved in proliferation of peroxisomes in yeast in response to oleate
Authors: EINERHAND Alexandra W. C. , KOS Wilko T. , DISTEL Ben , TABAK Henk F.
Published: March 2005

Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the proliferation of these organelles in Saccharomyces cerevisiae. Previously, we have reported the identification of a region containing a positive regulatory element in the 5′ flanking region of the FOX3 gene encoding the peroxisomal enzyme 3‐oxoacyl‐CoA thiolase. This region contains a 23‐bp imperfect inverted‐repeat sequence. Full induction, in response to oleate, is mediated by the intact dyad. However, one half‐site of the inverted repeat is also able to mediate induction of transcription in response to oleate, albeit to a small extent. Furthermore, the weak binding of protein to each part of the inverted repeat proved to be correlated with the weak activation of transcription, in support of oleate.

A DNase‐I footprint covered the entire dyad and DNA band‐shift experiments indicated that one or more trans‐acting factors bind to the imperfect palindrome. The binding of protein to this element seems to be correlated with transcriptional activation, since mutations in both halves of the inverted dyad affected both transcriptional activation and protein binding in vitro.

Similar oleate‐responsive elements are commonly found in the 5′ flanking regions of genes encoding proteins involved in peroxisome biogenesis and the factor(s) binding to oleate‐responsive element(s) could therefore be involved in coordination of the expression of oleate‐inducible genes and the proliferation of peroxisomes.
Sex pheromone plasmid pAD1‐encoded aggregation substance of Enterococcus faecalis is positively regulated in trans by traE1
Authors: MUSCHOLL Albrecht , GALLI Dominique , WANNER Gerhard , WIRTH Reinhard
Published: March 2005

Sex‐pheromone‐plasmid‐bearing strains of Enterococcus faecalis react with sex pheromone to induce the expression of an adhesin, the so‐called aggregation substance, on their cell surface. Here we show that, by complementation studies, for sex‐pheromone plasmid pAD1, expression of the structural gene asa1, coding for an aggregation substance, is mediated by a diffusible factor encoded on pAD1. We were able to demonstrate that a small open reading frame, traE1, is sufficient for transcription of the operon containing asa1. A model for expression of asa1 under the influence of the positive regulator is presented, which is supported by our observation that regulation involves an all‐or‐nothing induction phenomenon, leading to cells either fully expressing asa1 or not at all.
Structural features of the human gene for muscle‐specific enolase
Authors: GIALLONGO Agata , VENTURELLA Silvana , OLIVA Daniele , BARBIERI Giovanna , RUBINO Patrizia , FEO Salvatore
Published: March 2005

We report here the isolation and characterization of the human gene for the β or muscle‐specific isoform of the glycolytic enzyme enolase. The nucleotide sequence analysis revealed structural features, such as organization as 11 coding exons, the first exon consisting of an untranslated sequence and hence resembling sequences of the other two members of the gene family, the α and γ enolase genes. The β enolase locus spans about 6 kbp genomic DNA. Sequences matching the consensus sequence for muscle‐specific regulatory factors are present in the 5′‐flanking region and within the first intron. A combination of primer extension, S1 nuclease protection and RNA‐sequencing experiments indicates that the gene has a unique transcriptional start site, 26 bp downstream of a TATA‐like box; the differential usage of two donor sites within the untranslated exon I generates two alternatively spliced transcripts. The existence of the two mRNA, differing from one another in the presence or absence of a 42‐nucleotide fragment in the leader sequence, was confirmed by cloning the corresponding cDNA using the rapid amplification of cDNA ends strategy. Secondary‐structure predictions indicated that the leader sequences of the spliced forms could form hairpin structures with different free energies of formation, suggesting translational control.
The solution structure of the leucine zipper motif of the Jun oncoprotein homodimer
Authors: JUNIUS F. Keith , WEISS Anthony S. , KING Glenn F.
Published: March 2005

Proton NMR studies have been performed on a 9.8‐kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two‐dimensional scalar and dipolar‐coupling experiments enabled almost all proton resonances to be sequence‐specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled‐coil arrangement of parallel α‐helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen‐bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled‐coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled‐coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.
Amino acid sequence of the human intermediate basic protein 2 (HPI2) from sperm nuclei
Authors: ALIMI Elisabeth , MARTINAGE Arlette , ARKHIS Ahmed , BELAICHE Denise , SAUTIERE Pierre , CHEVAILLIER Philippe
Published: March 2005

Human intermediate basic protein 2 (HPI2) is a low‐molecular‐mass basic protein present in small amounts in human sperm nuclei. The amino acid composition of the protein, its N‐terminal amino acid sequence and peptide maps obtained after digestion with endoproteinases Lys‐C and Glu‐C, reveal that HPI2 is structurally related to human protamine species P2 (HP2), which is rich in Arg, His and Cys residues. Compared to HP2, which is one of the two major sperm protamines, HPI2 has an N‐terminal extension of 24 residues which includes six acidic residues and does not possess any Arg residues. The amino acid sequence of HPI2 (81 residues) is identical to the sequence of the C‐terminal region of another minor sperm nuclear protein, human intermediate basic protein 1 (HPI1, 101 residues), which was sequenced previously [Martinage, A., Arkhis, A., Alimi, E., Sautière, P. & Chevaillier, P. (1990) Eur. J. Biochem. 191, 449–451]. Due to this structural similarity, HPI2 must be considered as an intermediate in the maturation of proprotamine HPI1 limited proteolysis.
Purification and characterization of the major tyrosine protein kinase from the human promyelocytic cell line, HL60
Authors: ERNOULD Anne‐Pascale , FERRY Gilles , BARRET Jean‐Marc , GENTON Annie , BOUTIN Jean A.
Published: March 2005

The major tyrosine protein kinase from HL60 (a human non‐differentiated promyelocytic cell line) has been purified almost to homogeneity as judged by silver‐stained SDS/PAGE. The procedure involved four chromatographic steps: DEAE‐Sepharose, casein‐agarose, cibacron‐blue–agarose and hexyl‐agarose. The purification resulted in more than 1000‐fold enrichment in angiotensin II phosphorylation activity. A gel‐sizing experiment, labeling with [35S]ATP[γs] and autophosphorylation of the enzyme in the presence of [γ‐32P]ATP, all led to the identification of a single protein species with a molecular mass of about 40 kDa. Western blot experiments showed that this protein does not belong to the src family and is not related to the abl and fes oncogene products. Phosphorylation of angiotensin II and casein by this 40‐kDa human promyelocytic kinase was stimulated by high ionic strength especially from class IA metal salts. The Km for ATP was 2 μM and the Vmax 3.1 nmol · min−1· mg−1 using angiotensin II as a substrate. The kinase requires the presence of either Mn2+ or Mg2+ for full activity and utilizes ATP or dATP but not GTP as phosphate donor. Based on numerous biochemical observations, it was possible to demonstrate that kinase is different from any other tyrosine protein kinases described in the literature. This 40‐kDa protein was used as a molecular tool for testing some tyrosine protein kinase inhibitors described in the literature. It is one of the rare tyrosine protein kinases purified from human cancer cells to date.
Mitochondrial transport of mitoribosomal proteins, YmL8 and YmL20, in Saccharomyces cerevisiae
Authors: MATSUSHITA Yasuhiko , ISONO Katsumi
Published: March 2005

Two mitochondrial ribosomal (mitoribosomal) proteins, YmL8 and YmL20, of the yeast Saccharomyces cerevisiae and their derivatives were synthesized in vitro and their transport into isolated yeast mitochondria was examined. Of the two proteins, YmL20 possesses an N‐terminal presequence of 18 amino acid residues, while YmL8 has no such presequence. Both proteins were found to be transported into isolated mitochondria in an energy‐dependent manner. Furthermore, YmL20 protein without its N‐terminal presequence was also transported, despite the fact that the presequence alone was capable of transporting a fused passenger protein, Chinese hamster dihydrofolate reductase (DHFR). Therefore, YmL20 protein appears to possess redundant transport signals in its structure. Similarly, YmL8 derivatives lacking either 40 or 86 amino acid residues from the N‐terminus and/or 52 amino acid residues from the C‐terminus were transported. In addition, the N‐terminal segment of this protein was capable of transporting Chinese hamster DHFR into mitochondria, while its C‐terminal segment was not. Thus, YmL8 protein also appears to possess two or more transport signals in its structure. Perhaps the presence of many basic amino acid residues in these proteins might, at least partly, contribute to their mitochondrial transport.
Energy‐dependent changes in the conformation of the chloroplast ATP synthase and its catalytic activity
Authors: KOMATSU‐TAKAKI Mizuho
Published: March 2005

Chloroplast ATP synthase changes its conformation depending on the transmembrane electrochemical potential difference of protons (ΔμH+). This conformational change is observable by measuring the change in the reactivity of Lys109 of the ɛ subunit of choloroplast‐coupling‐factor 1. Illumination of thylakoids increased the ɛ‐Lys109 reactivity by a factor of 3–4 within 1 s. In the presence of ADP plus Pi, illumination of thylakoids increased the ɛ‐Lys109 reactivity by a factor of only 2. Addition of ATP in the post‐illumination dark or in the light after prior illumination increased the ɛ‐Lys109 reactivity depending on the concentration of coexisting NH4Cl. ATP hydrolysis at high level was observed irrespective of the ɛ‐Lys109 reactivity.
Si‐face stereospecificity at C5 of coenzyme F420 for F420‐dependent N5,N10‐methylenetetrahydromethanopterin dehydrogenase, F420‐dependent N5,N10‐methylenetetrahydromethanopterin reductase and F420H2:dimethylnaphthoquinone oxidoreductase
Authors: KUNOW Jasper , SCHWÖRER Beatrix , SETZKE Edgar , THAUER Rudolf K.
Published: March 2005

Coenzyme F420‐dependent enzymes catalyze the reversible reduction of F420 by stereospecific hydride transfer to C5 of 5‐deazaflavin. Two F420‐dependent enzymes have been investigated with respect to the stereochemistry of hydride transfer, the F420‐dependent NADP reductase and the F420‐reducing hydrogenase. Both enzymes were found to be Si‐face specific. In this study we report that three additional F420‐dependent enzymes are also Si‐face specific: N5,N10‐methylenetetrahydromethanopterin dehydrogenase, N5,N10‐methylenetetrahydromethanopterin reductase and coenzyme F420H2: dimethylnaphthoquinone oxidoreductase (F420H2 dehydrogenase). Thus, all five characterized F420‐dependent enzymes are Si‐face specific, which is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides and both Si‐face specific and Re‐face specific pyridine‐nucleotide‐dependent enzymes exist.