bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular analysis of Chs3p participation in chitin synthase III activity
Authors: Cos Teresa , Ford Richard A. , Trilla Jose Angel , Duran Angel , Cabib Enrico , Roncero Cesar
Published: March 2003

Chitin is a minor but essential component of the cell wall of Saccharomyces cerevisiae, with functions in septum formation in the vegetative life cycle and also in conjugation and spore cell‐wall synthesis in the sexual cycle. Of the three chitin synthases present in yeast, chitin synthase III (CSIII) is responsible for the synthesis of most of the chitin found in the cell, including a chitin ring at early budding, chitin interspersed in the cell wall, and chitin laid down during the sexual cycle. We have tagged Chs3p, the putative catalytic subunit of CSIII, with the immunoreactive epitope of influenza virus hemagglutinin to follow expression of the protein. Little correlation was found between the levels of transcription and translation of Chs3p and in vivo function, supporting our previous conclusion that regulation of CSIII occurs at the posttranslational level. To identify possible regions of the protein involved in catalysis or regulation, mutations were generated in the QRRRW ’signature sequence' of chitin synthases. Arginine residue mutations in Chs3p, and in Chs1p and Chs2p, resulted in a loss of both function in vivo and enzymatic activity. Mutations in a serine residue adjacent to glutamine in Chs3p caused loss of function in vivo with a moderate decrease in CSIII activity, suggesting a regulatory role for the serine residue in chitin biosynthesis. Several truncations in the unique hydrophilic carboxy‐terminal region of Chs3p identified a sequence of about 25 amino acids that is required for both function and in vitro activity. Since this region is not present in Chs1 or Chs2, it may be involved in the specific regulation of CSIII.
Butylated hydroxytoluene modulates DNA methylation in rats
Authors: Vanyushin Boris F. , Lopatina Nadezhda G. , Wise Carolyn K. , Fullerton Floyd R. , Poirier Lionel A.
Published: March 2003

The major observation of this investigation is that a single intraperitoneal injection of butylated hydroxytoluene (BHT, 60 mg/kg body mass) results within a few hours in a strong increase in nuclear DNA(cytosine‐5)‐methyl transferase (methyl transferase) activity in the liver, kidneys, heart, spleen, brain and lungs of male rats. In most organs, the rise in methyl transferase activity is observed as early as 4 h after BHT injection, it reaches a maximum at 8 h and then, except for lungs and brain, gradually decreases to its initial level at 16 h. At the maximum induction times, the methyl transferase activity in liver, kidney and spleen increases by about 16‐, 3‐ and 5‐fold, respectively. A second BHT injection at 96 h results in a secondary rise in hepatic methyl transferase activity. Isoelectric focusing electrophoresis of control rat liver nuclear extracts showed methyl transferase activity in the pI 4.7 and 7.4 protein fractions. Both fractions methylate calf thymus DNA better than they do Drosophila melanogaster DNA. In similar extracts from BHT‐treated rats, the methyl transferase activity is found in three protein fractions with pI values equal to 4.0, 6.2 and 9.5, respectively. Most of the methyl transferase fractions from the livers of BHT‐treated rats methylate the completely unmethylated D. melanogaster DNA better than they do calf thymus DNA. Thus, BHT induces methyl transferase activity that preferably provides de novo DNA methylation. BHT injection had no significant effect on the hepatic contents of S‐adenosylmethionine (AdoMet), S‐adenosylhomocysteine (AdoHcy) and AdoMet/AdoHcy ratios. While BHT injection did not alter the 5‐methyldeoxycytidine content in liver DNA, it did appear to alter such content in other organs. BHT appears to cause the reversible changes in the methylation status of an internal cytosine residue in some CCGG sites of the rat liver cytosine DNA‐methyl transferase gene. BHT induces also hypomethylation of the renal methyl transferase gene and the hepatic c‐Ha‐ras gene. While BHT also increases the hepatic mRNA transcripts for the S‐adenosylmethionine synthetase and the p53 genes, it had no detectable effects on the corresponding mRNA transcripts for methyl transferase homologous to murine methyl transferase. Thus, BHT induces tissue‐specific reversible changes in methyl transferase activity and methylation of total DNA and various genes in rats. A strong increase in methyl transferase activity in rat liver is accompanied with BHT‐induced change in the methyl transferase set observed in this organ.
Membrane‐bound proton‐translocating pyrophosphatase of Syntrophus gentianae, a syntrophically benzoate‐degrading fermenting bacterium
Authors: Schöcke Ludger , Schink Bernhard
Published: March 2003

Syntrophus gentianae is a strictly anaerobic bacterium which ferments benzoate to acetate, CO2 and H2 in the presence of hydrogen‐utilizing partner bacteria. Benzoate is activated by a benzoyl CoA ligase enzyme which forms AMP and pyrophosphate as coproducts. Pyrophosphatase activity was found to be largely membrane bound. Pyrophosphate hydrolysis was associated with proton translocation across the cytoplasmic membrane. Proton translocation could be abolished by the protonophor carbonylcyanide p‐chlorophenylhydrazone, and could also be coupled to ATP formation in membrane vesicle preparations. The ratio of ATP formation/pyrophosphate hydrolysis was 1 : 3. The reverse reaction, ATP‐dependent pyrophosphate synthesis, was possible with the same coupling stoichiometry. Pyrophosphatase was 90 % saturated at 1 mM pyrophosphate; pyrophosphate concentrations higher than 5 mM inhibited enzyme activity. Inhibition studies with ATP and EDTA indicated that MgPP.es−.rb.eii.rb was probably the physiological substrate. The optimum temperature was 35 +C. In the presence of Mg2+, the enzyme was remarkably heat stable, with 50 % of its maximum activity after 10 min at 60 +C. Exogenously added pyrophosphate could not be used for energy conservation.
Light regulates the rate of translation elongation of chloroplast reaction center protein D1
Authors: Edhofer Ingrid , Mühlbauer Stefan Klaus , Eichacker Lutz Andreas
Published: March 2003

Intact and lysed chloroplasts isolated from the day or night phase of seedling growth exhibit a higher rate of [35S]Met incorporation into the D1 protein in the light than in darkness. In the presence of the translation initiation inhibitor lincomycin, radiolabel incorporation remains unaffected for 7.5−10 min of the in vitro translation reaction, indicating that radiolabel incorporation is regulated by translation elongation. The rate of [35S]Met incorporation into D1‐protein can be increased by addition of exogenous ATP to the in vitro translation reactions; however, ATP cannot replace light, and at physiological concentrations of stromal ATP (40 μM), the rate is at least 25‐fold higher in the light than in darkness. This indicates that translation elongation is arrested in darkness. Separation of translation‐elongation reactions into polysome‐bound and membrane‐integrated D1 proteins demonstrates that the rate of translation elongation is higher in the presence of light. In the light, less time is required to transiently radiolabel a D1 translation intermediate of about 17 kDa and to chase the translation intermediate into mature D1 protein. We propose that light regulates the enzymatic activity of the translation‐elongation process in chloroplasts.
The chick and human collagen α1(XII) gene promoter
Authors: Chiquet Matthias , Mumenthaler Urs , Wittwer Matthias , Jin William , Koch Manuel
Published: March 2003

A single gene encodes collagen XII, an extracellular matrix protein with three large fibronectin‐related subunits connected via a short collagen triple helix. Since collagen XII is a component of a specific subset of collagen fibrils in tissues bearing high tensile stress, we are interested to know how its restricted expression is regulated. To this aim, we have isolated the region around the first exon of both the chick and human collagen α1(XII) gene. The upstream sequences of the two genes share common features but are not related. Strong similarity starts about 100 bp 5′ of the first exon and ends 100 bp into the first intron. In addition, two large conserved regions (56−63 % similarity) were found in the first intron. A single major and two clusters of minor transcription start sites were identified in both the chick and human gene. To test for promoter activity, conserved fragments from the chick gene were cloned into reporter plasmids for transient transfection of fibroblasts. A 70‐bp stretch containing a conserved nuclear factor‐1 binding sequence just upstream of the first transcription start site was found to work as a basal promoter. An adjacent, but nonoverlapping short segment including the more downstream start sites and a conserved TATTAA sequence exhibited independent promoter activity. GC‐rich sequences just 5′ and 3′ of the minimal promoter fragments were required for full activity. In contrast, inclusion of more upstream sequences (up to 2.4 kb) had no effect. The two conserved regions in the first intron showed no promoter activity on their own but modulated activity when linked to autologous or heterologous promoters. Specifically, one of these intronic regions might contain enhancer element(s) that respond to mechanical stress acting on the fibroblasts. We conclude that the collagen XII gene is driven by a basal promoter with two halves that can act independently; conserved control regions are located around the first exon and in the first intron.
UDP‐galactose 4‐epimerase from Kluyveromyces fragilis : Reconstitution of holoenzyme structure after dissociation with parachloromercuribenzoate
Authors: Majumdar Siddhartha , Bhattacharjee Hiranmoy , Bhattacharyya Debasish , Bhaduri Amarnath
Published: March 2003

UDP‐galactose 4‐epimerase from yeast Kluyveromyces fragilis (Kluyveromyces marxianus var. marxianus) is a homodimer of molecular mass 75 kDa/subunit and has one mol NAD firmly bound/dimer. The pathway for the assembly of the holoenzyme structure has been studied after dissociating the native epimerase with p‐chloromercuribenzoate into inactive mercurated monomers. The process of dissociation was not associated with unfolding of the molecules. Reconstitution of the functional holoenzyme was done by reduction with dithiothreitol and addition of extra NAD. The reaction was thus followed to monitor maturation of the enzyme from the folded monomeric state. The reconstituted enzyme was similar to the native enzyme in terms of a number of physiochemical properties such as secondary, tertiary and quarternary structures, Km for the substrate UDP‐galactose, reductive inhibition, interaction with the fluorophore 1‐anilino 8‐naphthalene sulphonic acid (ANS), etc. Reconstitution under low ionic strength buffer (I = 0.011) shows that the presence of NAD is essential for the formation of a dimeric structure. However, dimeric apoenzyme could also be stabilized under high ionic strength buffer (I = 0.1). Reactivation was strongly dependent on pH, being most effective at pH 8.1. Kinetic evidence suggested that, at low ionic strength, assembly of NAD over dimeric apoenzyme is the rate‐limiting step in expressing catalytic activity. This process has a low energy of activation of 27.2 kJ/mol.
Evidence for existence of multiple conformations of kinesin and ncd motor domains in solution revealed by 31P‐NMR of the tightly bound ADP
Authors: Suzuki Yoshikazu , Tanokura Masaru , Shimizu Takashi
Published: March 2003

The motor domains of kinesin and ncd contain ADP bound tightly at the enzymatic active sites even after purification. The microenvironments surrounding the phosphates of the tightly bound ADP molecules in these motor domains were studied using 31P‐NMR. The α‐phosphate and β‐phosphate of the tightly bound ADP molecules gave 31P‐NMR signals at positions shifted to higher and lower field, respectively, from those of free MgADP in solution. On the other hand, the peak areas of the signals of the α‐phosphates and β‐phosphates of bound ADP were much smaller than those of free MgADP. The temperature dependence of these signals was investigated to reveal that each signal of bound MgADP exhibited a large temperature dependence in the peak area from 0 to 20 +C with both proteins. This result implied that these motor domains would take multiple conformations in solution; one of these conformations gives the signals of bound MgADP observed by 31P‐NMR as above, while another makes the signals of MgADP very broad and, hence, invisible in the 31P‐NMR spectra. A possible reason for invisible signals would be strong chemical shift anisotropy effects of the phosphorus nuclei. In addition, we found a difference between kinesin and ncd motor domains in the chemical shift of the α‐phosphate of bound MgADP, indicating that the electrostatic or magnetic microenvironments of this site of these motor domains differed from each other.
Molecular cloning and tissue distribution of rat sarcosine dehydrogenase
Authors: Bergeron Francois , Otto Annegret , Blache Philippe , Day Robert , Denoroy Luc , Brandsch Roderich , Bataille Dominique
Published: March 2003

Sarcosine dehydrogenase (SarDH) is a mitochondrial flavoenzyme involved in the oxidative degradation of choline to glycine. The absence of SarDH activity in humans is genetically transmitted and is the cause of an amino acid metabolism disorder called sarcosinemia. Tryptic fragments of the purified enzyme from rat liver were subjected to Edman degradation and the sequences obtained were used to clone the cDNA encoding the full length protein. The deduced amino acid sequence of SarDH shares an overall similarity of 47 % with dimethylglycine dehydrogenase (Me2GlyDH), another flavoenzyme involved in the mitochondrial choline catabolism with a similar FAD‐binding domain. Covalent binding of FAD to SarDH was demonstrated by the observation of strong fluorescence at 530 nm under excitation at 450 nm of the enzyme immunoprecipitated under denaturing conditions from liver extracts. The localization of SarDH immunoreactivity in the mitochondrial matrix was confirmed by Western‐blot analysis of purified mitochondrial fractions. Finally, the tissue distribution of SarDH was investigated by Northern‐blot analysis of total RNA and Western‐blot analysis of total protein from several rat tissues. A strong expression in the liver, but also in the lung, pancreas, kidney, thymus, and oviduct was observed. We therefore suggest that the enzymes of the choline catabolism pathway are important also for metabolism in nonhepatic tissues.
Kinetic studies on the activation of dopamine β‐monooxygenase by copper and vanadium ions
Authors: Abudu Ntei , Banjaw Mehret Y. , Ljones Torbjørn
Published: March 2003

Dopamine β‐monooxygenase requires copper ions for catalytic activity. The stoichiometry of copper activation has been a matter of discussion, but most of the recent literature agrees on a model with two copper ions per active site. We have now reinvestigated this problem with kinetic experiments at high and low protein levels. The apoenzyme (metal free) is rapidly activated by adding copper. Incremental addition of copper to high levels (up to 10 μM subunits) of enzyme raised the catalytic activity until the stoichiometric relationship between copper and enzyme subunits was 1 : 1. No increase in activity was observed upon addition of copper in excess of this up to levels of 3 Cu/subunit. Experiments at low protein levels (0.12 μM subunits) revealed that copper activation is described by a hyperbolic, Michaelis‐Menten‐type curve. This is to be expected for the 1 Cu/subunit model, whereas the 2/1 model predicts sigmoid curves. With an incremental addition of apoenzyme (high level) to a fixed level of copper, a sharp break was again observed at 1 Cu/subunit, and excess apoenzyme showed no evidence of the inhibition predicted by the 2 Cu/subunit model. Steady‐state kinetics experiments with variation of the concentrations of copper and the three substrates supported an equilibrium‐ordered mechanism, whereby a single activating copper ion is trapped in the active site by the substrates. Treatment of enzyme containing more than 1 Cu/subunit [both the Cu(I) and Cu(II) states were examined] with a chelating column resulted in loss of all copper in excess of 1 Cu/subunit. Reactivation of apoenzyme by vanadyl ions was studied, both as dopamine β‐monooxygenase‐catalyzed electron transfer and hydroxylation. The maximal velocity with vanadium was 70 % of that with copper, and the activation curve was clearly hyperbolic, again supporting the requirement of only one metal ion per active site. In conclusion, our results support the view that the copper ions bound to dopamine β‐monooxygenase in excess of 1 Cu/subunit are not required for catalysis.
Characterization of cytochrome c‐556 from the purple phototrophic bacterium Rhodobacter capsulatus as a cytochrome‐c peroxidase
Authors: Hu Wei , De Smet Lina , Van Driessche Gonzalez , Bartsch Robert G. , Meyer Terrance E. , Cusanovich Michael A. , Van Beeumen Jozef
Published: March 2003

A cytochrome c‐556 was purified from Rhodobacter capsulatus and the complete amino acid sequence was determined. It contains 328 amino acid residues and two typical heme‐binding sites at cysteine residues 54 and 57 and at residues 200 and 203. It is homologous to the family of bacterial cytochrome c peroxidases (BCCP) with 69 % identity to Paracoccus denitrificans BCCP and 60 % identity to Pseudomonas aeruginosa BCCP for which there is a three‐dimensional structure. There is lesser similarity to the mauG gene products from methylotrophic bacteria which are thought to be involved in biosynthesis of the quinone cofactor of methylamine dehydrogenase. Translated genes from Escherichia coli and Helicobacter pylori are also related to the bacterial cytochrome c peroxidases. The divergence of this family of proteins is reflected in the fact that the reported sixth heme ligands are not conserved, except in Pseudomonas, Rhodobacter and Paracoccus. This suggests that homologs of BCCP may fold differently and/or may not have the same enzymatic activity as the prototypic protein from Ps. aeruginosa. We found that the Rb. capsulatus BCCP is active with both Rb. capsulatus cytochrome c2 and with horse cytochrome c as substrates (Km values 60 μm and 6 μm, respectively). The turnover number was 40 s−1 and the Km for peroxide was 33 μm. We have thus confirmed that the Rb. capsulatus protein is a cytochrome c peroxidase.
Human matrix metalloproteinase‐9 : activation by limited trypsin treatment and generation of monoclonal antibodies specific for the activated form
Authors: Duncan Margaret E. , Richardson Jonathan P. , Murray Graeme I. , Melvin William T. , Fothergill John E.
Published: March 2003

For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and activated forms of the enzymes. Activated human matrix metalloproteinase‐9 (MMP‐9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N‐terminal sequencing. Trypsin treatment led to a stepwise removal of the propeptide domain and also caused cleavage within the C‐terminal domain. Monoclonal antibodies specific for the activated form of human MMP‐9 were raised by using a peptide corresponding to the N‐terminus of the activated enzyme as immunogen. The antibodies do not recognise the MMP‐9 proenzyme or the active or proenzyme forms of matrix metalloproteinase‐2 (MMP‐2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP‐9 in formalin‐fixed, wax‐embedded sections from a series of oesophageal cancer cases previously shown to contain MMP‐9. All of the tumours contained activated MMP‐9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin‐fixed, wax‐embedded sections, they should prove useful for the detection of activated MMP‐9 in various disease processes.
The gene for the Alzheimer‐associated β‐amyloid‐binding protein (ERAB) is differentially expressed in the testicular Leydig cells of the azoospermic by w/wv mouse
Authors: Hansis Christoph , Jähner Detlev , Spiess Andrej‐Nikolai , Boettcher Kay , Ivell Richard
Published: March 2003

In order to discover possible new testicular paracrine factors involved in the establishment of spermatogenesis, a modified differential display reverse transcription, polymerase chain reaction (DDRT‐PCR) procedure was used to detect gene transcripts preferentially expressed in the testes of the azoospermic w/wv mutant mouse. One of the differentially expressed gene products showed partial similarity to members of the short‐chain alcohol dehydrogenase family of enzymes. This cDNA fragment was used to obtain the full‐length mouse cDNA sequence, which initially showed moderate similarity to a 20β‐steroid dehydrogenase from lower organisms, and later shown to have > 85 % similarity to a novel endoplasmic‐reticulum‐associated‐binding protein (ERAB) from the human brain, implicated in Alzheimer's disease. A recently cloned bovine sequence also of high similarity suggests that this molecule might also represent an isozyme of 3‐hydroxyacyl‐CoA dehydrogenase. Using the mouse cDNA as probe, northern hybridization showed enrichment of the transcript to the testicular Leydig cells, and showed a specific approximately 20‐fold enrichment in the azoospermic mouse testis. The level of the testicular ERAB transcript does not seem to change through puberty, suggesting that a lack of germ cells alone is not responsible for the up‐regulation in the w/wv testis. Using the three‐dimensional coordinates of the published 20β‐hydroxysteroid dehydrogenase structure as template, it was additionally possible to construct a molecular model of the novel protein which showed it to have a very similar structure to this enzyme, including the substrate‐binding domain.
Determinants of the fidelity of processing glucoamylase‐lysozyme fusions by Aspergillus niger
Authors: Spencer J. Andrew , Jeenes David J. , Mackenzie Donald A. , Haynie Donald T. , Archer David B.
Published: March 2003

Fusion proteins are used to enhance the yields of heterologous proteins secreted from filamentous fungi. In Aspergillus niger, the target protein is normally fused downstream of the carrier protein glucoamylase with a Lys‐Arg KEX2‐like cleavage site at the junction. This is cleaved in vivo to release mature protein but the processing is not always accurate. We have used N‐terminal mutant lysozymes to vary the sequence immediately downstream of the KEX site, and also varied the amino acid sequence upstream of the KEX processing site, to study the fidelity of processing. The sequences both upstream and downstream of the KEX2 site affected the fidelity of cleavage. With some constructs, a range of processing sites were apparent and the relative proportions were time dependent in batch cultures of A. niger. Aberrant processing was related to the secondary‐structure preferences of the amino acids in and around the KEX site. Downstream of the processing site, the fidelity of processing decreased in proportion to the tendency for helix formation.
Rapid analysis of epitope‐paratope interactions between HIV‐1 and a 17‐amino‐acid neutralizing microantibody by electrospray ionization mass spectrometry
Authors: Millar Alan L. , Jackson Nicholas A. C. , Dalton Howard , Jennings Keith R. , Levi Michael , Wahren Britta , Dimmock Nigel J.
Published: March 2003

Progress in therapeutic or prophylactic immune intervention in HIV‐1 infections may only come about with a detailed understanding at the molecular/atomic level of how antibodies neutralize (inactivate) virus infectivity. Currently information on the molecular aspects of antibody‐virus interaction comes predominantly from X‐ray crystallography, a process that is dependent on the production of suitable crystals [1]. NMR can also be valuable [2] but is complex and time consuming, while mass spectrometry has been limited to matrix‐assisted laser‐desorption ionization (MALDI) analysis of peptides eluted from the cognate antibody [3]. Here, we have used electrospray ionization mass spectrometry (ESI‐MS) to detect directly the interactions of a novel 17‐amino‐acid microantibody (MicroAb) that has HIV‐1‐inhibitory activity [4], and peptides representing the V3 regions of primary HIV‐1 strains isolated from Brazil (clade B) and Africa (clade A). The MicroAb is based on the third complementarity‐determining region of the heavy chain (CDR‐H3) of a murine monoclonal IGGI (F58) specific for the V3 loop of the gp120 envelope glycoprotein of HIV‐1. ESI‐MS proved to be rapid (taking < 3 h for the entire analysis), sensitive (analytes at 2 mmol/ml), and accurate (RMM estimation to 0.01−0.1 %). With it, we showed that the MicroAb forms complexes with the V3 peptides, implying that its antiviral activity is mediated by binding directly to the virus particle. In addition, through controlled protease digestion of the V3 peptides, we concluded that the CDR‐H3 MicroAb bound to RKXXXIGPGR, a region similar to the epitope of the whole IgG as determined by ELISA. We believe that the approach exemplified here will be applicable generally to the identification of groups involved in receptor‐ligand interactions.
Progress curves
Authors: Nutt Anu , Sild Veljo , Pettersson Göran , Johansson Gunnar
Published: March 2003

The chain‐end preference and processivity of the cellulases 1,4‐β‐ D‐glucan‐cellobiohydrolase I (CBH I) and 1,4‐β‐
D‐glucan‐cellobiohydrolase II (CBH II) from Trichoderma reesei and 1,4‐β‐
D‐glucan‐cellobiohydrolase 50 (CBH 50) and 1,4‐β‐
D‐glucan‐cellobiohydrolase 58 (CBH 58) from Phanerochaete chrysosporium were studied by comparing experimental degradation data on reducing end‐labelled bacterial microcrystalline cellulose with computer simulations of different models. Our results with T. reesei and P. chrysosporium cellulases show that there is a common pattern of hydrolysis for CBH‐I‐type enzymes and another clearly distinguishable pattern for CBH‐II‐type enzymes, especially in the initial part of the progress curve.
Site‐directed mutagenesis studies of the metal‐binding center of the iron‐dependent propanediol oxidoreductase from Escherichia coli
Authors: Obradors Núria , Cabiscol Elisa , Aguilar Juan , Ros Joaquim
Published: March 2003

The amino acid residues involved in the metal‐binding site in the iron‐containing dehydrogenase family were characterized by the site‐directed mutagenesis of selected candidate residues of propanediol oxidoreductase from Escherichia coli. Based on the findings that mutations H263R, H267A and H277A resulted in iron‐deficient propanediol oxidoreductases without catalytic activity, we identified three conserved His residues as iron ligands, which also bind zinc. The Cys362, a residue highly conserved among these dehydrogenases, was considered another possible ligand by comparison with the sequences of the medium‐chain dehydrogenases. Mutation of Cys362 to Ile, resulted in an active enzyme that was still able to bind iron, with minor changes in the Km values and decreased thermal stability. Furthermore, in an attempt to produce an enzyme specific only for the zinc ion, three mutations were designed to mimic the catalytic zinc‐binding site of the medium‐chain dehydrogenases : (1) V262C produced an enzyme with altered kinetic parameters which nevertheless retained a significant ability to bind both metals, (2) the double mutant V262C−M265D was inactive and too unstable to allow purification, and (3) the insertion of a cysteine at position 263 resulted in a catalytically inactive enzyme without iron‐binding capacity, while retaining the ability to bind zinc. This mutation could represent a conceivable model of one of the steps in the evolution from iron to zinc‐dependent dehydrogenases.
Papaya glutamine cyclase, a plant enzyme highly resistant to proteolysis, adopts an all‐β conformation
Authors: Oberg Keith A. , Ruysschaert Jean‐Marie , Azarkan Mohamed , Smolders Nicole , Zerhouni Samira , Wintjens René , Amrani Amina , Looze Yvan
Published: March 2003

Glutamine cyclases catalyse the conversion of L‐glutaminyl‐peptides into 5‐oxoprolyl‐peptides with the concomitant liberation of ammonia. We report here biophysical characterisation of the glutamine cyclase present in the laticiferous cells of the plant Carica papaya. After purification to near homogeneity, this enzyme was subjected to limited proteolysis and found to exhibit a high resistance to degradation and nicking. The structural reasons for this property were examined using circular dichroism and infrared spectroscopies. By combining the analyses of the infrared and CD spectra of papaya glutamine cyclase, its susceptibility to proteolysis, and its hydrogen‐deuterium exchange characteristics, we conclude that this protein contains extensive β‐sheet structure and is likely to have only short immobile loops connecting its β‐strands.
Cloning and biochemical characterisation of Aspergillus niger hexokinase
Authors: Panneman Henk , Ruijter George J. G. , van den Broeck Hetty C. , Visser Jaap
Published: March 2003

The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54 090, a pI of 4.9 and is a homodimer.
D‐Glucose, the glucose analogue 2‐deoxy‐
D‐glucose,
D‐fructose,
D‐mannose and
D‐glucosamine are phosphorylated by hexokinase, whereas the hexoses
D‐galactose,
L‐sorbose, methyl α‐
D‐glucoside and the pentoses
L‐arabinose and
D‐xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1−0.2 mM) of trehalose 6‐phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6‐phosphate is competitive towards the sugar substrate (Ki = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3−4 h after depletion of the carbon source.
cDNA cloning, in vitro expression, and biochemical characterization of cholinesterase 1 and cholinesterase 2 from amphioxus
Authors: McClellan James Scott , Coblentz William Blake , Sapp Mathew , Rulewicz Gabriel , Gaines David Isaac , Hawkins Ashley , Ozment Caroline , Bearden Amy , Merritt Sarah , Cunningham John , Palmer Elizabeth , Contractor Amir , Pezzementi Leo
Published: March 2003

We have isolated cDNAs coding for the complete amino acid sequences of cholinesterase 1 (ChE1) and cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts have the characteristics of H‐type catalytic subunits, which are inserted in the membrane via an ethanolamine‐glycan‐phosphatidylinositol anchor. The members of the catalytic triad of ChEs, the three pairs of cysteine residues involved in intrachain disulfide bonding, a cysteine near the carboxy terminal of both sequences, which could mediate interchain disulfide bonding, and 11 of the 14 aromatic amino acids that line the catalytic gorge of AChE are conserved. A remarkable difference between the two enzymes is in the region of the acyl‐binding pocket, which plays an important role in determining substrate specificity in cholinesterases. ChE2 contains a sequence that resembles the acyl pocket of invertebrate ChE, while the acyl‐binding site of ChE1 is novel. There are also differences between the two enzymes in the peripheral anionic site, which mediates inhibition by certain ligands. In vitro expression in COS‐7 cells demonstrates that ChE2 hydrolyzes acetylthiocholine almost exclusively, while ChE1 hydrolyzes both acetylthiocholine and butyrylthiocholine. Both enzymes are inhibited comparably by BW284c51, but ChE1 is considerably more resistant to inhibition by propidium, ethopropazine, and eserine than is ChE2. Velocity sedimentation indicates that ChE1 and ChE2 are present as amphiphilic and nonamphiphilic G2 forms in vivo and in vitro. Another molecular form, which sediments at 17 S, is also present in vivo. Nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol‐specific phospholipase C demonstrates that the vast majority of ChE1 and ChE2 is present as ethanolamine‐glycan‐phosphatidylinositol‐anchored G2 forms in vivo. ChE1 also possesses an ethanolamine‐glycan‐phosphatidylinositol‐anchor in vitro; however, ChE2 produced in vitro could not be detected on nondenaturing gels.
Solution structure of the recombinant oxidized rabbit uteroglobin using homonuclear and heteronuclear multidimensional NMR
Authors: Winkelmann Rolf , Geschwindner Stefan , Haun Matthias , Rüterjans Heinz
Published: March 2003

Rabbit uteroglobin (rab‐UG) is a 16‐kDa homodimeric secretory protein with potent anti‐inflammatory/immunomodulatory properties. Its physiological role is still unclear, although it was observed that several small hydrophobic molecules bind to the oxidized and the reduced uteroglobin. It is suggested that the formation and/or disruption of the two disulphide bridges not only regulates this binding itself, but also the affinity to the ligand. The determination of the solution structure has been started with the assignment of 1H, 15N and 13C resonances of the oxidized rabbit uteroglobin, based on several two‐dimensional and three‐dimensional homonuclear and heteronuclear double and triple resonance experiments. The assignment was possible with the overproduction of the wild‐type as well as of uniformly 15N‐labeled and 15N/13C‐labeled samples of the recombinant protein. A complete assignment of 1H, 15N and 13C resonances, the secondary‐structure elements and the tertiary structure in solution is presented. The tertiary solution structure was found to be in good agreement with the previously determined crystal structure of rab‐UG and with the solution structure of human uteroglobin (h‐UG). h‐UG and rab‐UG are extremely stable proteins within a wide range of pH and temperatures. Some of the binding characteristics of ligands of rab‐UG and a mutant with all cysteine residues exchanged to serine residues are discussed.
Structure determination for octasaccharides derived from the carbohydrate−protein linkage region of chondroitin sulphate chains in the proteoglycan aggrecan from bovine articular cartilage
Authors: Huckerby Thomas N. , Lauder Robert M. , Nieduszynski Ian A.
Published: March 2003

Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6‐year‐old to 8‐year‐old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by β‐elimination. The individual oligosaccharides were purified by strong anion‐exchange chromatography and their structures determined by very high‐field one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat‐region extensions to the core tetrasaccharide linkage region structure. They have the following structures :

ΔUA(β1−3)GalNAc(β1−4)GlcA(β1−3)GalNAc(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)GalNAc(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol
and
ΔUA(β1−3)GalNAc4S(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol.

They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide‐repeat‐region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6‐sulphated. However, in a significant amount, only one of the residues was 6‐sulphated while the other was either unsulphated or 4‐sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.
Effects of histone and diolein on the structure of phosphatidylcholine/phosphatidylserine or phosphatidylcholine/phosphatidylglycerol bilayers
Authors: Goldberg Edward M. , Borchardt Dan B. , Zidovetzki Raphael
Published: March 2003

The effects of the PKC substrate histone 1 and the PKC activator diolein (Ole2Gro) on the structure of phosphatidylcholine (PtdCho)/phosphatidylserine (PtdSer), or PtdCho/phosphatidylglycerol (PtdGro) bilayers were studied using 2H‐NMR. The results showed that in PtdCho/PtdSer bilayers, histone preferentially increased order parameters of the acyl chains of the PtdSer, but not the PtdCho lipid component. This effect was additive with the effect of Ole2Gro, which equally increased the ordering of the acyl chains of both PtdCho and PtdSer. The histone‐induced change in the conformation of the PtdCho headgroups in PtdCho/PtdSer bilayers indicated that positively charged residues of the bound histone are located above the lipid−water interface and their location was altered by the presence of Ole2Gro. A different picture was observed in the case of PtdCho/PtdGro bilayers; although the effect of Ole2Gro on both the PtdCho or the PtdGro components was similar to the case of the PtdCho/PtdSer bilayers, histone did not significantly affect the order parameters of PtdCho or PtdGro in either the absence or presence of Ole2Gro. The results indicate that histone 1 induces clustering of PtdSer in PtdCho bilayers which may contribute to PKC activation. Moreover, the observed differences in the interactions of histone with PtdCho/PtdSer compared with PtdCho/PtdGro bilayers may explain the higher efficiency of PtdSer in activating PKC.
DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus : Processivity and fidelity of misinsertion and mispair extension
Authors: Taube Ran , Avidan Orna , Bakhanashvili Mary , Hizi Amnon
Published: March 2003

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B‐type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579−587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3′ end of the nascent DNA strand and the ability to extend 3′ mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.
Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and ζ‐carotene desaturase, expressed in Escherichia coli, catalyze a poly‐cis pathway to yield pro‐lycopene
Authors: Bartley Glenn E. , Scolnik Pablo A. , Beyer Peter
Published: March 2003

We have expressed in Escerichia coli the enzymes geranylgeranyl diphosphate synthase and phytoene synthase, from the soil bacterium Erwinia stewartii, and the two carotene desaturases phytoene desaturase and carotene ζ‐carotene desaturase from Arabidopsis thaliana. We show that pro‐lycopene (7,9,7′,9′‐tetra‐cis)‐lycopene is the main end product of the plant desaturation pathway in these cells. In addition, light is required in this system. Whereas in the dark mainly ζ‐carotene, the phytoene desaturase product, accumulates, illumination leads to activation of this intermediate caused by its photoisomerization. ζ‐Carotene then meets the stereospecific requirements of ζ‐carotene desaturase and pro‐lycopene is formed. In contrast, a strain of E. coli carrying geranylgeranyl diphosphate synthase, phytoene desaturase and the bacterial carotene desaturase CrtI, which mediates lycopene formation from phytoene, does not require light, nor is a poly‐cis‐lycopene species formed. The stereoselectivity of the plant‐type desaturation pathway expressed in E. coli is the same as previously shown with chromoplast membranes. As the phytoene desaturase and ζ‐carotene desaturase used originate from a system not capable of developing chromoplasts, this indicates that the poly‐cis pathway of carotene desaturation may have a wider occurrence than initially believed.
1‐Methyl‐4‐phenylpyridinium ion (MPP+) selectively inhibits the replication of mitochondrial DNA
Authors: Miyako Kenichi , Irie Takashi , Muta Tsuyoshi , Umeda Shuyo , Kai Yoichiro , Fujiwara Toshiyuki , Takeshige Koichiro , Kang Dongchon
Published: March 2003

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine is known to cause Parkinsonism in its neurotoxic form, 1‐methyl‐4‐phenylpyridinium ion (MPP+). We have previously reported that MPP+ decreases the content of mitochondrial DNA (mtDNA) independently of the inhibition of complex I in human cells [Miyako, K., Kai, Y., Irie, T., Takeshige, K., and Kang, D. (1997) J. Biol. Chem.272, 9605–9608]. Here we study the mechanism causing the decrease in mtDNA. MPP+ inhibits the incorporation of 5‐bromo‐2′‐deoxyuridine into mtDNA but not into nuclear DNA, indicating that MPP+ inhibits the replication of mtDNA but not that of the nuclear genome. The replication of mtDNA is initiated by the synthesis of the heavy strand switched from the transcription of the light strand. MPP+ decreases the nascent heavy strands per mtDNA and increases the transcript of the ND6 gene, encoded on light strand, per mtDNA. The amount of mitochondrial transcription factor A is not decreased. These data suggest that the transcription is not inhibited and therefore the transition from transcription to replication of mtDNA is lowered in the MPP+‐treated cells. Electron microscopy shows that the number of mitochondria is not decreased in the MPP+‐treated cells, suggesting that MPP+ does not affect the overall biogenesis of mitochondria. Thus, MPP+ selectively inhibits the replication of mtDNA.
Synergism between a half‐site and an imperfect estrogen‐responsive element, and cooperation with COUP‐TFI are required for estrogen receptor (ER) to achieve a maximal estrogen‐stimulation of rainbow trout ER gene
Authors: Petit Fabrice G. , Métivier , Raphaël , Valotaire , Yves , Pakdel Farzad
Published: March 2003

In all oviparous, liver represents one of the main E2‐target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up‐regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within – 40/– 248 enhancer region. Absence of interference with steroid hormone receptors and tissue‐specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis‐acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen‐responsive element (ERE) and a consensus half‐ERE to achieve a high hormone‐dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter‐transcription factor I (COUP‐TFI) through enhancing autoregulation. Using a point mutation COUP‐TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP‐TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP‐TFI requires specifically the AF‐1 transactivation function of ER and can be observed in the presence of E2 or 4‐hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone‐responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis‐elements and/or trans‐acting factors, such as ER itself or COUP‐TF.