bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural diversity and transcription of class III peroxidases from Arabidopsis thaliana
Authors: Welinder Karen G. , Justesen Annemarie F. , Kjærsgård Inger V. H. , Jensen Rikke B. , Rasmussen Søren K. , Jespersen Hans M. , Duroux Laurent
Published: March 2003

Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) encoding novel heme‐containing class III peroxidases from Arabidopsis thaliana and annotated 73 full‐length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ‐specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene‐specific and highly sensitive reverse transcriptase‐polymerase chain reaction (RT‐PCR).

We predicted that 71 genes could yield stable proteins folded similarly to horseradish peroxidase (HRP). The putative mature peroxidases derived from these genes showed 28–94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N‐terminal signal peptides. In 20 peroxidases these signal peptides were followed by various N‐terminal extensions of unknown function which are not present in HRP. Ten peroxidases showed a C‐terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III peroxidases accounted for an impressive 2.2% of root ESTs. Rather few peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse peroxidases (< 70% sequence identity). Furthermore, genes appearing in tandem showed distinct expression profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous peroxidases in other plant species and will provide a common platform for combining knowledge of peroxidase structure and function relationships obtained in various species.
The Ikaros family protein Eos associates with C‐terminal‐binding protein corepressors
Authors: Perdomo José , Crossley Merlin
Published: March 2003

Eos is a zinc finger transcription factor of the Ikaros family. It binds typical GGGAA Ikaros recognition sites in DNA and functions as a transcriptional repressor. Here we show that Eos associates with the corepressor C‐terminal‐binding protein (CtBP). CtBP has previously been shown to bind Pro‐X‐Asp‐Leu‐Ser (PXDLS) motifs in several DNA‐binding proteins. We note that Eos contains a related motif PEDLA, and we demonstrate that CtBP can bind this site weakly but that it also contacts additional regions of Eos. Consistent with this finding, mutation of the PEDLA motif does not negate CtBP binding or CtBP‐mediated repression by Eos. CtBP has previously been shown to bind to a PXDLS‐type motif in Ikaros, and we show that another Ikaros‐related protein TRPS1 also contains a PXDLS CtBP contact motif within its repression domain. We conclude that several Ikaros family proteins utilize CtBP corepressors to inhibit gene expression.
Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration
Authors: Itoh Hideaki , Komatsuda Atsushi , Ohtani Hiroshi , Wakui Hideki , Imai Hirokazu , Sawada Ken‐ichi , Otaka Michiro , Ogura Masahito , Suzuki Akira , Hamada Fumio
Published: March 2003

There are few reports concerning the sorting mechanisms of mammalian HSP60 into the mitochondria from the cytoplasm. In the present study we investigated the protein import system. Based on immunoblotting and immuno‐histochemistry, HSP60 was detected in both the cytoplasm and mitochondria. The purified cytoplasmic HSP60 showed chaperone activity, and the protein was imported into the mitochondria in vitro by a mitochondrial import assay. HSP60 mRNA was increased in the kidney papilla of rats that had been water restricted for three and five days, but no changes in HSP60 mRNA were detected in the cortex or the medulla of the rat kidneys. Upon immunoblotting, HSP60 was detected in both the cytoplasm and the mitochondria of normal rat kidney cortex, medulla, and papilla in almost the same quantity. HSP60 was remarkably decreased in the kidney papilla of rats that were water restricted but the protein was increased in the mitochondria of the rat kidney papilla. We also analysed binding of the protein to the signal sequence of HSP60 using signal sequence‐affinity column chromatography. We identified only one protein band with a molecular mass of 70 kDa on SDS/PAGE. The protein was eluted from the affinity column by an excess of signal peptide or by 5 mm ATP. Upon immunoblotting, the 70‐kDa protein cross‐reacted with an antibody against HSP70. These results suggested that mammalian HSP60 is located both in the cytoplasm as a stable cytoplasmic HSP60 and also in the mitochondria under normal conditions. The cytoplasmic HSP60 is quickly imported into the mitochondria under severe conditions by cytoplasmic HSP70.
Guanosine diphosphate‐4‐keto‐6‐deoxy‐d‐mannose reductase in the pathway for the synthesis of GDP‐6‐deoxy‐d‐talose in Actinobacillus actinomycetemcomitans
Authors: Suzuki Nao , Nakano Yoshio , Yoshida Yasuo , Nezu Takashi , Terada Yoshihiro , Yamashita Yoshihisa , Koga Toshihiko
Published: March 2003

The serotype a‐specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6‐deoxy‐d‐talose. Guanosine diphosphate (GDP)‐6‐deoxy‐d‐talose is the activated sugar nucleotide form of 6‐deoxy‐d‐talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP‐6‐deoxy‐d‐talose synthetic enzymes, GDP‐α‐d‐mannose 4,6‐dehydratase and GDP‐4‐keto‐6‐deoxy‐d‐mannose reductase, in the gene cluster required for the biosynthesis of serotype a‐specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed‐phase HPLC (RP‐HPLC). The sugar nucleotide produced from GDP‐α‐d‐mannose by these enzymes was purified by RP‐HPLC and identified by electrospray ionization‐MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP‐6‐deoxy‐d‐talose is produced from GDP‐α‐d‐mannose. This paper is the first report on the GDP‐6‐deoxy‐d‐talose biosynthetic pathway and the role of GDP‐4‐keto‐6‐deoxy‐d‐mannose reductase in the synthesis of GDP‐6‐deoxy‐d‐talose.
Expression and characterization of recombinant vitamin K‐dependent γ‐glutamyl carboxylase from an invertebrate, Conus textile
Authors: Czerwiec Eva , Begley Gail S. , Bronstein Mila , Stenflo Johan , Taylor Kevin , Furie Barbara C. , Furie Bruce
Published: March 2003

The marine snail Conus is the sole invertebrate wherein both the vitamin K‐dependent carboxylase and its product, γ‐carboxyglutamic acid, have been identified. To examine its biosynthesis of γ‐carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811‐amino‐acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and ≈ 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K‐dependent carboxylase activity and vitamin K‐dependent epoxidase activity. The recombinant carboxylase has a molecular mass of ≈ 130 kDa. The recombinant Conus carboxylase carboxylated Phe‐Leu‐Glu‐Glu‐Leu and the 28‐residue peptides based on residues −18 to +10 of human proprothrombin and proFactor IX with Km values of 420 µm, 1.7 µm and 6 µm, respectively; the Km for vitamin K is 52 µm. The Km values for peptides based on the sequence of the conotoxin ε‐TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 µm, 75 µm and 74 µm, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide‐binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K‐dependent γ‐carboxylases argue for conservation of a vitamin K‐dependent carboxylase across animal species and the importance of γ‐carboxyglutamic acid synthesis in diverse biological systems.
Relaxin‐like bioactivity of ovine Insulin 3 (INSL3) analogues
Authors: Claasz Antonia A. , Bond Courtney P. , Bathgate Ross A. , Otvos Laszlo , Dawson Nicola F. , Summers Roger J. , Tregear Geoffrey W. , Wade John D.
Published: March 2003

Relaxin is an insulin‐like peptide consisting of two separate chains (A and B) joined by two inter‐ and one intrachain disulfide bonds. Binding to its receptor requires an Arg–X–X–X–Arg–X–X–Ile motif in the B‐chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg–X–X–X–Arg motif within its B‐chain, although this is displaced by four amino acids towards the C‐terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin‐like activity although analogue (Analogue A) with an introduced arginine residue in the B‐chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichrosim spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin‐like activity. These results indicate that while the arginine cassette is vital for relaxin‐like activity, there are additional, as yet unidentified structural requirements for relaxin binding.
Expression of MsPG3‐GFP fusions in Medicago truncatula‘hairy roots’ reveals preferential tip localization of the protein in root hairs
Authors: Rodríguez‐Llorente Ignacio D. , Pérez‐Hormaeche Javier , Dary Mohammed , Caviedes Miguel A. , Kondorosi Adam , Ratet Pascal , Palomares Antonio J.
Published: March 2003

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle‐enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3‐GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3‐GFP fusions were expressed in Medicago truncatula‘hairy roots’ following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3‐GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.
New insights into the P‐glycoprotein‐mediated effluxes of rhodamines
Authors: Loetchutinat Chatchanok , Saengkhae Chantarawan , Marbeuf‐Gueye Carole , Garnier‐Suillerot Arlette
Published: March 2003

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma drug transporter P‐glycoprotein (P‐gp). This protein is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Despite the advances of recent years, we still have an unclear view of the molecular mechanism by which P‐gp transports such a wide diversity of compounds across the membrane. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. The aim of the present study was to further characterize the transport of several rhodamine analogues, either positively charged or zwitterionic. We took advantage of the intrinsic fluorescence of rhodamines and performed a flow‐cytometric analysis of dye accumulation in the wild‐type drug sensitive K562 that do not express P‐gp and its MDR subline that display high levels of MDR. The measurements were made in real time using intact cells. The kinetic parameter, ka = VM/km, which is a measure of the efficiency of the P‐gp‐mediated efflux of a substrate was similar for almost all the rhodamine analogues tested. In addition these values were compared with those determined previously for the P‐gp‐mediated efflux of anthracycline. Our conclusion is that the compounds of these two classes of molecules, anthracyclines and rhodamines, are substrates of P‐gp and that their pumping rates at limiting low substrate concentration are similar. The findings presented here are the first to show quantitative information about the kinetic parameters for P‐gp‐mediated efflux of rhodamine analogues in intact cells.
Selecting cells with different Alzheimer's disease γ‐secretase activity using FACS
Authors: Sernee M. Fleur , Evin Geneviève , Culvenor Janetta G. , Villadangos José A. , Beyreuther Konrad , Masters Colin L. , Cappai Roberto
Published: March 2003

The ultimate step in Alzheimer's disease Aβ generation involves γ‐secretase, which releases Aβ from its membrane‐bound precursor. A similar presenilin‐dependent proteolytic activity is implicated in the release of the Notch intracellular domain. We have developed a novel assay for γ‐secretase activity based on green fluorescent protein detection. This involves cotransfection of a substrate‐activator based on the amyloid precursor protein or the Notch sequence and a fluorescent reporter gene. Stable fluorescent cell populations were selected by fluorescent activated cell sorting and characterized. This assay enabled the identification and sorting of populations, which differ in their levels of γ‐secretase activity, with high fluorescent cells producing more Aβ than low fluorescent cells. Specific γ‐secretase inhibitors, L‐685,458 and MW167, reduced cell fluorescence in a dose‐dependent manner that paralleled inhibition of Aβ secretion. Overexpression of presenilin 1 increased the cell fluorescence. Cells expressing presenilin with different aspartate mutations (D257A, D385A and D257A/D385A) or exon 9 deletion mutation showed reduced fluorescence. The single aspartate mutations showed a concomitant reduction in Aβ secretion, whereas the D257A/D385A and ΔE9 mutations had no effect on Aβ secretion.
Cofactor‐independent oxygenation reactions catalyzed by soluble methane monooxygenase at the surface of a modified gold electrode
Authors: Astier Yann , Balendra Suki , Hill H. Allen O. , Smith Thomas J. , Dalton Howard
Published: March 2003

Soluble methane monooxygenase (sMMO) is a three‐component enzyme that catalyses dioxygen‐ and NAD(P)H‐dependent oxygenation of methane and numerous other substrates. Oxygenation occurs at the binuclear iron active centre in the hydroxylase component (MMOH), to which electrons are passed from NAD(P)H via the reductase component (MMOR), along a pathway that is facilitated and controlled by the third component, protein B (MMOB). We previously demonstrated that electrons could be passed to MMOH from a hexapeptide‐modified gold electrode and thus cyclic voltammetry could be used to measure the redox potentials of the MMOH active site. Here we have shown that the reduction current is enhanced by the presence of catalase or if the reaction is performed in a flow‐cell, probably because oxygen is reduced to hydrogen peroxide, by MMOH at the electrode surface and the hydrogen peroxide then inactivates the enzyme unless removed by catalase or a continuous flow of solution. Hydrogen peroxide production appears to be inhibited by MMOB, suggesting that MMOB is controlling the flow of electrons to MMOH as it does in the presence of MMOR and NAD(P)H. Most importantly, in the presence of MMOB and catalase, the electrode‐associated MMOH oxygenates acetonitrile to cyanoaldehyde and methane to methanol. Thus the electochemically driven sMMO showed the same catalytic activity and regulation by MMOB as the natural NAD(P)H‐driven reaction and may have the potential for development into an economic, NAD(P)H‐independent oxygenation catalyst. The significance of the production of hydrogen peroxide, which is not usually observed with the NAD(P)H‐driven system, is also discussed.
Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites
Authors: Kaiser Liselotte , Gafvelin Guro , Johansson Eva , van Hage‐Hamsten Marianne , Rasool Omid
Published: March 2003

We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long‐term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon‐γ and interleukin‐5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release.
Insights into the design of a hybrid system between Anabaena ferredoxin‐NADP+ reductase and bovine adrenodoxin
Authors: Faro Merche , Schiffler Burkhard , Heinz Achim , Nogués Isabel , Medina Milagros , Bernhardt Rita , Gómez‐Moreno Carlos
Published: March 2003

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin‐NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin‐NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin‐NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild‐type) ferredoxin‐NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin‐NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.
Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single‐tryptophan mutants
Authors: Pattanaik Priyaranjan , Ravindra Gudihal , Sengupta Chandana , Maithal Kapil , Balaram Padmanabhan , Balaram Hemalatha
Published: March 2003

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active‐site loop 6. Two single‐tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild‐type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue‐shifted emission (321 nm) and exhibits significant red‐edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1‐Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue‐shifted emission from a tryptophan in a polar environment in the protein. The wild‐type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2‐phosphoglycolic acid.
pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger
Authors: Pařenicová Lucie , Benen Jacques A. E. , Kester Harry C. M. , Visser Jaap
Published: March 2003

In the present study, the molecular and basic biochemical characterization of endopolygalacturonase E, the fourth Aspergillus niger N400 endopolygalacturonase, is reported. The entire endopolygalacturonase E gene consists of 1293 bp interrupted by three short introns (50, 50, and 59 bp, respectively) as concluded from the cDNA sequence. The deduced amino acid sequence comprises 378 residues that include 39 N‐terminal amino acids of the prepropeptide. The calculated Mr and pI of the mature protein are 35 584 and 3.6, respectively.

Compared with other endopolygalacturonases from A. niger N400, the mature protein endopolygalacturonase E has the highest sequence identity with endopolygalacturonase C (77.6 %) followed by endopolygalacturonase I (57.6 %) and endopolygalacturonase II (54.3 %).

For overproduction of endopolygalacturonase E, an A. niger multicopy strain was used that was transformed with a promoter gene fusion construct that directs expression from the glycolytic A. niger pyruvate kinase promoter. The enzyme was purified and characterized as an endopolygalacturonase based on product analysis after polygalacturonate hydrolysis and on bond cleavage frequencies of oligogalacturonates of different degree of polymerisation (n = 2−7). The pH optimum was 3.8. The Km and Vmax for polygalacturonate hydrolysis were 2.5 ± 0.4 mg ml−1 and 1.3 ± 0.2 μkat mg−1, respectively. A subsite map was calculated by the combination of the methods of Suganuma et al. [Suganuma, T., Matsuno, R., Ohnishi, M. & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 293−316] and Nitta et al. [Nitta, Y., Mizushima, M., Hiromi, K. & Ono, S. (1971) J. Biochem. (Tokyo) 69, 567−576]. This indicated that the enzyme was composed of at least five subsites.
Topology of subunit a of the Escherichia coli ATP synthase
Authors: Jäger Heike , Birkenhäger Ralf , Stalz Wolf‐Dieter , Altendorf Karlheinz , Deckers‐Hebestreit Gabriele
Published: March 2003

The antigenic determinants of mAbs against subunit a of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic decapeptides. For two of the mAbs the epitopes are E4NMTPQD10 (GDH 14‐5C6) and V29DPQ32 (GDH 8‐8B3). Binding of these mAbs to membrane vesicles of different orientation revealed that both epitopes are accessible in vesicles with inside‐out orientation. These results demonstrate that at least the N‐terminal amino acids 1−32 of subunit a are located at the cytoplasmic side of the membrane. A further determination of the topology of subunit a was performed by inserting the reporter epitope DYKDDDDK (FLAG epitope) at different positions of the polypeptide chain. 10 of 13 insertions led to a functional F0F1 ATP synthase and allowed specific detection of the modified subunit a by immunoblotting using an mAb against the FLAG epitope. In addition, polyclonal anti‐FLAG IgG was applied for the recognition of the mutant FLAG epitope DYKDDVDK. Cells carrying this mutant FLAG epitope at the C terminus of subunit a were able to grow on succinate as sole carbon and energy source, revealing a functional ATP synthase, in contrast to those carrying the original FLAG epitope at the same position. Binding studies with membrane vesicles of different orientation and anti‐FLAG Ig demonstrated that both termini of the protein are located at the cytoplasmic side of the membrane, indicating that an even number of membrane‐spanning segments is present in subunit a. In addition, insertion of two FLAG epitopes in tandem after K66, or one epitope after H95, and Q181 revealed that the polypeptide regions including these residues are accessible from the cytoplasmic surface of the membrane. These results support the view that the polypeptide chain of subunit a traverses the membrane six times.
Multidrug‐resistance drug‐binding peptides generated by using a phage display library
Authors: Popkov Mikhail , Lussier Isabelle , Medvedkine Viatcheslav , Estève Pierre‐Olivier , Alakhov Valery , Mandeville Rosemonde
Published: March 2003

A phage display library of random decapeptides was used to generate peptide ligands that can bind multidrug‐resistance (MDR) drugs mimicking, in this respect, the drug‐binding activity of P‐glycoprotein. Seven peptide sequences were identified that specifically bound doxorubicin. Five of these sequences expressed the core consensus motif WXXW. The displacement assay showed that the phages expressing these peptides bound MDR type drugs (vinblastine, doxorubicin, verapamil, and genistein) with the same selectivity as P‐glycoprotein and did not interact with non‐MDR type drugs, such as arabinosylcytosine (Ara‐C) and melphalan. One of the selected peptides that showed a highest capacity for the binding (VCDWWGWGIC) was synthesized and displayed competition with the phage for doxorubicin binding. The structure modeling suggested that all the selected sequences contained a hydrophobic envelope in which MDR drugs could be docked with substantial energy minimization. Western blot analysis showed that monospecific antibody obtained against the phage expressing VCDWWGWGIC peptide could specifically recognize P‐glycoprotein in the membrane fraction of MDR phenotype MCF‐7ADR cells. The MDR drug‐binding sequences generated during this work could provide an important tool for design and screening of new chemotherapeutic agents.
Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II
Authors: Levitsky Dmitrii I. , Ponomarev Michael A. , Geeves Michael A. , Shnyrov Valery L. , Manstein Dietmar J.
Published: March 2003

The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry. These fragments M754 and M761 correspond to the globular motor portion of the myosin head that contains ATP‐ and actin‐binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761 : the maximum of the thermal transition occurred at 41.7 +C for M754 and at 45.6 +C for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755−761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2−3.5 +C shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate, beryllium fluoride or aluminium fluoride causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of beryllium fluoride was less distinct than that of aluminium fluoride or orthovanadate. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by differential scanning calorimetry in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head.
GTP‐binding protein γ12 subunit phosphorylation by protein kinase C
Authors: Asano Tomiko , Morishita Rika , Ueda Hiroshi , Asano Masahisa , Kato Kanefusa
Published: March 2003

We have demonstrated previously that the GTP‐binding protein γ12 subunit is a selective substrate for phosphorylation by protein kinase C among various γ subunits in vitro, and that a serine residue in the N‐terminal region is involved. In the present study, we first determined that the site of phosphorylation was Ser1 with antibodies developed against two N‐terminal peptides containing phosphorylated Ser1 and Ser2, respectively. Using an antibody recognizing phosphorylated γ12 and Swiss 3T3 cells rich in this protein, γ12 was found to be phosphorylated by stimulation of quiescent cells with various reagents, such as phorbol 12‐myristate 13‐acetate (PMA), NaF, fetal calf serum, lysophosphatidic acid, endothelin, and growth factors. Pertussis toxin completely and partially prevented phosphorylation of γ12 induced by lysophosphatidic acid and fetal calf serum and by endothelin, respectively, suggesting a contribution of Gi/o. Phosphorylation of γ12 was limited when cells were stimulated by a single reagent, even with PMA, a strong activator of protein kinase C, whereas simultaneous stimulation with lysophosphatidic acid and either PMA or platelet‐derived growth factor induced a synergistic increase of phosphorylation, suggesting physiological roles for GTP‐binding proteins and protein kinase C in combination. Phosphorylated γ12 was also detected in various tissues of untreated rats. Its decrease by pertussis toxin treatment also suggested the involvement of Gi/oin vivo.
Calmodulin antagonists increase the expression of membrane‐type‐1 matrix metalloproteinase in human uterine cervical fibroblasts
Authors: Ito Akira , Yamada Miwako , Sato Takashi , Sanekata Kyoko , Sato Hiroshi , Seiki Motoharu , Nagase Hideaki , Mori Yo
Published: March 2003

The treatment of human uterine cervical fibroblasts with concanavalin A (ConA), or a specific calmodulin antagonist, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7) or trifluoperazine resulted in accumulation of an active form of matrix metalloproteinase 2 (MMP‐2, gelatinase A). In contrast, N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide (W‐5), a weaker antagonist of calmodulin, did not modulate the activation of proMMP‐2. The activation of proMMP‐2 was confirmed by the enhanced activity on gelatin and the conversion of proMMP‐2 to a 62‐kDa form by zymography and western blotting. The plasma membrane, but not the conditioned medium, of the W‐7‐ or trifluoperazine‐treated cells activated proMMP‐2; this activation was blocked by membrane‐type‐1 MMP (MT1‐MMP) antibody and EDTA. The plasma membrane from trifluoperazine‐ or ConA‐treated cells contained MT1‐MMP and tissue inhibitor of metalloproteinases 2. Both trifluoperazine treatment and ConA treatment increased the steady‐state levels of MT1‐MMP mRNA and proMMP‐2 mRNA. These results, together with our previous observations on the production of proMMP‐1 (interstitial procollagenase) and proMMP‐3 (prostromelysin 1) [Ito, A., Sato, T., Ojima, Y., Chen, L.‐C., Nagase, H. & Mori, Y. (1991) J. Biol. Chem. 266, 13 598−13 601], suggest that calmodulin negatively regulates the matrix turnover by suppressing the production of a number of proMMPs including proMMP‐1, proMMP‐3 and MT1‐MMP, and the activation of proMMP‐2 in human uterine cervical fibroblasts.
Deletion and site‐directed mutagenesis of the Wolinella succinogenes fumarate reductase operon
Authors: Simon Jörg , Gross Roland , Ringel Michael , Schmidt Enrico , Kröger Achim
Published: March 2003

Wolinella succinogenes is known to grow at the expense of fumarate respiration with formate or sulfide as electron donor. A W. succinogenes mutant (ΔfrdCAB) lacking the fumarate reductase operon did not grow with fumarate as terminal electron acceptor and either formate or sulfide as electron donor. The ΔfrdCAB mutant grown with formate and nitrate did not contain fumarate reductase activity and did not catalyze electron transport from sulfide to fumarate, in contrast to the nitrate‐grown wild‐type strain. A mutant constructed by integration of frdCAB into the ΔfrdCAB mutant genome showed wild‐type properties with respect to growth and enzyme activities. The frdC2 gene located downstream of the fumarate reductase operon frdCAB possibly encodes a diheme cytochrome b that is similar to FrdC (41 % identical residues). The corresponding transcript differs from that of frdCAB. A ΔfrdC2 mutant showed wild‐type properties with respect to growth and enzyme activities. Using site‐directed mutagenesis, each of the four histidine residues that are predicted to serve as the axial heme ligands in FrdC (His44, His93, His143, and His182) was replaced by alanine or other residues. The resulting mutants did not grow with formate and fumarate and did not contain fumarate reductase activity, FrdA or FrdC when grown with formate and nitrate. In contrast, substitution of two histidine residues that are not considered heme ligands, yielded mutants (H114A and H120A) that grew with fumarate.

It is concluded that FrdCAB is an obligatory component of fumarate respiration with formate and with sulfide in W. succinogenes. FrdC2 is not involved in fumarate respiration. Replacement of the putative heme ligands of FrdC prevents formation of a functional fumarate reductase.
Kinetic studies of allosteric catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa
Authors: Sainz Germaine , Tricot Catherine , Foray Marie‐Françoise , Marion Dominique , Dideberg Otto , Stalon Victor
Published: March 2003

The pseudo‐reverse reaction of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase in which arsenate is first coupled to citrulline followed by elimination of carbamylarsenate has been studied. Arsenate and citrulline saturation curves are sigmoidal. The different responsiveness of the transcarbamoylase to isosteric and allosteric ligands was examined both in the forward reaction, the carbamoylation of ornithine, and in the pseudo‐reverse reaction, the arsenolytic cleavage of citrulline. Nucleoside monophosphates and polyamines that act as allosteric activators and inhibitors, respectively, on the carbamoylation reaction have the same effect on the rate of the arsenolytic cleavage of citrulline. ATP and other nucleoside triphosphates were found to stimulate enzyme activity at low carbamoylphosphate concentration with little influence on the carbamoylphosphate concentration at half‐maximum velocity as well as on the cooperative index. When measuring the initial rate of the reverse reaction, the arsenolytic cleavage of citrulline, ATP was found to be a weak inhibitor, whereas CTP still stimulates the reaction and UTP was without influence. This unidirectional inhibition or activation phenomenon is likely apparent since initial studies were conducted and no consideration was given to equilibrium conditions. Regulation of catabolic OTCase by nucleoside triphosphates is without physiological meaning. In contrast, stimulation by nucleoside monophosphates may indicate that energy limitation could promote the synthesis and activity of the catabolic enzyme.
Structural determination of the O‐antigenic polysaccharide from the enterotoxigenic Escherichia coli O147
Authors: Hygge Blakeman Karin , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigenic polysaccharide from enterotoxigenic Escherichia coli O147 has been determined by NMR spectroscopy, and component and methylation analyses. The sequence of the sugar residues could be determined by NOESY and heteronuclear‐multiple‐bond‐connectivity NMR experiments. It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure :

←4)‐β‐
D‐GalpA‐(1←3)‐β‐
D‐GalpNAc‐(1←2)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1← ,
where Rha represents 6‐deoxymannose. The O‐antigen of E. coli O147 is identical to the repeating unit of Shigella flexneri serotype 6 lipopolysaccharide, except that the latter contains an O‐acetyl group at C3 of the rhamnosyl residue substituted by the N‐acetylgalactosamine residue. Immunochemical analyses using a monoclonal antibody specific for the S. flexneri serotype 6 O‐antigen showed an identical reactivity with both lipopolysaccharides.
The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection
Authors: Gamonet Franck , Lauquin Guy J.‐M.
Published: March 2003

Saccharomyces cerevisiae Lys7p was proposed to be the enzyme catalyzing the dehydratation of homocitrate to cis‐homoaconitate, the second step of the lysine biosynthetic pathway. In this communication we provide evidence that Lys7p is involved in oxidative stress protection. Cells deleted for the LYS7 gene displayed, in addition to lysine auxotrophy, methionine auxotrophy, sensitivity to superoxide generating drugs and light irradiation, and diminution of calcineurin activity. The SOD1 gene encoding the Cu/Zn‐superoxide dismutase was expressed in strains lacking Lys7p, and although Sod1p was produced in normal amounts no detectable enzyme activity was found. In contrast, the mitochondrial Mn‐superoxide dismutase activity did not seem to be impaired. lys7 cells exhibited a normal uptake of Cu from growth medium. The Cu/Zn‐superoxide dismutase activity was restored by addition of Cu (but not by addition of other metallic cations) to the growth medium or to cellular extracts, suggesting a lack of Cu2+ at the active site. These results render it necessary to reconsider the role of the Lys7p. Its involvement in Cu metabolism and oxidative‐stress protection, and the possibility of a human equivalent in amyotrophic lateral sclerosis are discussed.
Identification of the lamina‐associated‐polypeptide‐2‐binding domain of B‐type lamin
Authors: Furukawa Kazuhiro , Kondo Takao
Published: March 2003

Lamina‐associated polypeptide (LAP)2, which directly interacts with B‐type lamins and chromosomes, is an integral membrane protein specifically distributed along the inner nuclear membrane of the nuclear envelope. Multiple regions of its large nucleoplasmic domain promote this localization, including the first (residues 1−296) and the second (residues 298−409) halves of the LAP2 N terminus. The second half is involved in LAP2 association with the nuclear lamina [Furukawa, K., Panté, N., Aebi, U. & Gerace, L. (1995) EMBO J. 14, 1626−1636]. In this study to further define its role, we examined which domain of B‐type lamin interacts with LAP2 by means of a binding assay with bacterially expressed proteins and a yeast two‐hybrid system. We found that amino acids in the region of residues 78−258 of the lamin B1 rod domain directly bound with LAP2. The data suggest that LAP2 may modulate the assembly of nuclear lamins.
Engineering a change in metal‐ion specificity of the iron‐dependent superoxide dismutase from Mycobacterium tuberculosis
Authors: Bunting Karen , Cooper Jon. B. , Badasso Mohammed O. , Tickle Ian J. , Newton Melanie , Wood Steve P. , Zhang Ying , Young Douglas
Published: March 2003

We have refined the X‐ray structures of two site‐directed mutants of the iron‐dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal‐ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active‐site interactions since its side‐chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 Å of the metal‐binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X‐ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 Å and 2.5 Å, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three‐dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active‐site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.
The Flavobacterium okeanokoites adenine‐N6‐specific DNA‐methyltransferase M.FokI is a tandem enzyme of two independent domains with very different kinetic properties
Authors: Leismann Oliver , Roth Markus , Friedrich Tatjana , Wende Wolfgang , Jeltsch Albert
Published: March 2003

The Flavobacterium okeanokoites adenine‐N6‐specific DNA‐methyltransferase, M.FokI, modifies both adenine residues within its asymmetric recognition sequence 5′‐GGATG/CATCC‐3′. It is a fusion protein comprising two independent enzymes. We have cloned, overexpressed and purified full‐length M.FokI as well as both individual domains and analyzed their kinetics of DNA methylation using unmethylated and hemimethylated oligodeoxynucleotide substrates. Our data show that both domains of M.FokI methylate DNA independently of each other but cooperate in DNA binding. In agreement to former studies, the N‐terminal domain of M.FokI modifies the upper strand of the recognition sequence (GGATG). It strongly prefers hemimethylated (5′‐GGATG/CmATCC‐3′; mA = N6‐methyladenosine) over unmethylated substrates. In contrast, the C‐terminal domain prefers unmethylated DNA substrates. Surprisingly, in addition to methylating the lower strand of the recognition sequence (CATCC), M.FokI‐(335−647) also modifies the upper strand (GGATG), albeit with a lower activity. In addition, methylation was detected at CACCC sites, but not at sites in which a central AT dinucleotide is flanked by at least one A T or T A base pair. These results suggests that M.FokI‐(335−647) either has a very degenerate specificity for (G/C)AT(G/C) and sites similar to CATCC or GGATG or, alternatively, that it has a dual specificity for CATCC and GGATG.
A core‐shell model of calcium phosphate nanoclusters stabilized by β‐casein phosphopeptides, derived from sedimentation equilibrium and small‐angle X‐ray and neutron‐scattering measurements
Authors: Holt Carl , Timmins Peter A. , Errington Neil , Leaver Jeffrey
Published: March 2003

Calcium phosphate nanoclusters were prepared under standardised conditions using 10 mg ml−1 of the 25‐amino‐acid N‐terminal tryptic phosphopeptide of bovine β‐casein as a stabilising agent. The Mr determined by sedimentation equilibrium was 197 600 ± 13 700 and the apparent radius of gyration determined by X‐ray scattering was 2.80 ± 0.05 nm. A small‐angle neutron scattering contrast variation study in 1H2O/2H2O mixtures was performed and gave radii of gyration at the calculated match points for the calcium phosphate (88.2 %2H2O) and phosphopeptide (41.3 %2H2O) of 3.39 ± 0.08 nm and 1.85 ± 0.05 nm, respectively. Measurements at larger scattering wave vector showed a subsidiary maximum at about Q = 1.6 nm−1.

The results are consistent with a model of the nanoclusters comprising a spherical core of 355 ± 20 CaHPO4 2 H2O units, density 2.31 g ml−1 and radius 2.30 ± 0.05 nm surrounded by 49 ± 4 peptide chains with a partial specific volume of 0.7 cm3 g−1, forming a tightly packed shell with an outer radius of 4.04 ± 0.15 nm.

This model suggests that the phosphopeptide is able to arrest the process of growth of the precipitating phase of calcium phosphate at its earliest stages. A similar role for whole casein could be vital to the normal functioning of the mammary gland during milk secretion.