bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and bacterial expression of a general odorant‐binding protein from the cabbage armyworm Mamestra brassicae
Authors: Maibèche‐Coisne Martine , Longhi Sonia , Jacquin‐Joly Emmanuelle , Brunel Carole , Egloff Marie‐Pierre , Gastinel Louis , Cambillau Christian , Tegoni Mariella , Nagnan‐Le Meillour Patricia
Published: March 2003

A cDNA clone encoding a general odorant‐binding protein (GOBP2) was isolated from antennal RNA of Mamestra brassicae by reverse transcription‐PCR (RT‐PCR) and RACE‐PCR. The cDNA encoding the GOBP2 was further used for bacterial expression. Most of the recombinant GOBP2 (> 90 %) was found to be insoluble. Purification under denaturing conditions consisted of solubilisation of inclusion bodies, affinity chromatography, refolding and gel filtration. The refolded rGOBP2 was cross‐reactive with a serum raised against the GOBP2 of the Lepidoptera Antheraea polyphemus. The purified refolded rGOBP2 was further characterised by native PAGE, IEF, N‐terminal sequencing, and two‐dimensional NMR. A functional characterisation of the rGOBP2 was carried out by testing its ability to bind pheromone compounds. The yields of production and purification fulfil the requirements of structural studies.
Structure/function relationship of CYP11B1 associated with Dahl's salt‐resistant rats
Authors: Nonaka Yasuki , Fujii Tomomi , Kagawa Norio , Waterman Michael R. , Takemori Hiroshi , Okamoto Mitsuhiro
Published: March 2003

Dahl's salt‐resistant normotensive rats (DR rats) have been previously reported to express cytochrome P‐450 (CYP11B1) containing five missense mutations [Matsukawa, N., Nonaka, Y., Higaki, J., Nagano, M., Mikami, H., Ogihara, T. & Okamoto, M. (1993) J. Biol. Chem. 268, 9117−9121]. To investigate structure‐function relationships of CYP11B, wild‐type rat CYP11B1 and CYP11B2 and DR‐CYP11B1 (mutant CYP11B1 in Dahl's salt‐resistant rats) have been successfully expressed in Escherichia coli. Steroid 11β‐hydroxylase (11β‐OHase) activity observed with DR‐CYP11B1 was similar to that of wild‐type CYP11B1, while 18‐hydroxylase (18‐OHase) activity of DR‐CYP11B1 was lower than that of wild‐type CYP11B1. Mutant CYP11B1s containing a single or a double amino acid substitution associated with DR‐CYP11B1 have been also expressed in E. coli to investigate effects of the substitutions on enzymatic activity. Each of the single mutant enzymes showed lower 18‐OHase activity than wild‐type CYP11B1, but not as low as DR‐CYP11B1. A double mutant CYP11B1 with V381L and I384L showed 18‐OHase activity at a similar low level to that of DR‐CYP11B1. The 19‐hydroxylation (19‐OHase) activity of DR‐CYP11B1 was about one‐third of that of the wild‐type enzyme and this low activity appeared due to the V443M mutation. These results suggest that three of five amino acid substitutions present in DR‐CYP11B1 account for the decreased 18‐OHase and 19‐OHase activities. A decrease in these enzyme activities may be responsible for the normotension of the DR rats when fed a high‐salt diet.
Thrombopoietin potentiates the protein‐kinase‐C‐mediated activation of mitogen‐activated protein kinase/ERK kinases and extracellular signal‐regulated kinases in human platelets
Authors: Ezumi Yasuharu , Uchiyama Takashi , Takayama Hiroshi
Published: March 2003

The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal‐regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC‐dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen‐activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf‐1 and directly augmented the PKC‐mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf‐1. The MEK inhibitor PD098059 failed to affect not only thrombin‐induced or phorbol ester‐induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent‐soluble/non‐cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.
Transferrins
Authors: Bou Abdallah Fadi , El Hage Chahine Jean‐Michel
Published: March 2003

Fe(III) uptake by the iron‐delivery and iron‐scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo‐ovotransferrin loses two protons with K1a = 50 ± 1 nM and K2a = 4.0 ± 0.1 nM. These acid‐base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 ± 0.15 mM and KN = 110 ± 10 mM. FeNAc3 exchanges its Fe(III) with the C‐site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 ± 25 M−1 s−1, reverse rate constant k−1 = (6.0 ± 0.1)×103 M−1 s−1 and equilibrium constant K1 = 0.11 ± 0.01. This iron‐protein intermediate loses then a single proton, K3a = 3.50 ± 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 ± 0.01 s−1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 ± 0.05)×10−2 s−1. These modifications in the monoferric protein conformation are essential for iron uptake by the N‐site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15 000 s.

The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics ?
Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase
Authors: Henriksson Gunnar , Nutt Anu , Henriksson Hongbin , Pettersson Bert , Ståhlberg Jerry , Johansson Gunnar , Pettersson Göran
Published: March 2003

A 28‐kDa endoglucanase was isolated from the culture filtrate of Phanerochaete chrysosporium strain K3 and named EG 28. It degrades carboxymethylated cellulose and amorphous cellulose, and to a lesser degree xylan and mannan but not microcrystalline cellulose (Avicel). EG 28 is unusual among cellulases from aerobic fungi, in that it appears to lack a cellulose‐binding domain and does not bind to crystalline cellulose. The enzyme is efficient at releasing short fibres from filter paper and mechanical pulp, and acts synergistically with cellobiohydrolases. Its mode of degrading filter paper appears to be different to that of endoglucanase I from Trichoderma reesei. Furthermore, EG 28 releases colour from stained cellulose beads faster than any other enzyme tested. Peptide mapping suggests that it is not a fragment of another known endoglucanases from P. chrysosporium and peptide sequences indicate that it belongs to family 12 of the glycosyl hydrolases. EG 28 is glycosylated. The biological function of the enzyme is discussed, and it is hypothesized that it is homologous to EG III in Trichoderma reesei and the role of the enzyme is to make the cellulose in wood more accessible to other cellulases.
Biochemical characterization, molecular cloning and expression of laccases – a divergent gene family – in poplar
Authors: Ranocha Philippe , McDougall Gordon , Hawkins Simon , Sterjiades Raja , Borderies Gisèle , Stewart Derek , Cabanes‐Macheteau Marion , Boudet Alain‐M. , Goffner Deborah
Published: March 2003

The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2‐independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90‐kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm.

 The cDNA clones corresponding to the 90‐kDa and 110‐kDa proteins, lac90 and lac110, were obtained by a PCR‐based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene‐specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
Solution structure of a lipid transfer protein extracted from rice seeds
Authors: Poznanski Jarek , Sodano Patrick , Suh Se Won , Lee Jae Young , Ptak Marius , Vovelle Françoise
Published: March 2003

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three dimensional structure of rice nonspecific lipid transfer protein (ns‐LTP), a 91 amino acid residue protein belonging to the broad family of plant ns‐LTP. Sequence specific assignment was obtained for all but three HN backbone 1H resonances and for more than 95% of the 1H side‐chain resonances using a combination of 1H 2D NOESY; TOCSY and COSY experiments at 293 K. The structure was calculated on the basis of four disulfide bridge restraints, 1259 distance constraints derived from 1H‐1H Overhauser effects, 72 φ angle restraints and 32 hydrogen‐bond restraints. The final solution structure involves four helices (H1: Cys3‐Arg18, H2: Ala25‐Ala37, H3: Thr41‐Ala54 and H4: Ala66‐Cys73) followed by a long C‐terminal tail (T) with no observable regular structure. N‐capping residues (Thr2, Ser24, Thr40), whose side‐chain oxygen atoms are involved in hydrogen bonds with i + 3 amide proton additionally stabilize the N termini of the first three helices. The fourth helix involving Pro residues display a mixture of α and 310 conformation. The rms deviation of 14 final structures with respect to the average structure is 1.14 ± 0.16 Å for all heavy atoms (C, N, O and S) and 0.72 ± 0.01 Å for the backbone atoms. The global fold of rice ns‐LTP is close to the previously published structures of wheat, barley and maize ns‐LTPs exhibiting nearly identical pattern of the numerous sequence specific interactions. As reported previously for different four‐helix topology proteins, hydrophobic, hydrogen bonding and electrostatic mechanisms of fold stabilization were found for the rice ns‐LTP. The sequential alignment of 36 ns‐LTP primary structures strongly suggests that there is a uniform pattern of specific long‐range interactions (in terms of sequence), which stabilize the fold of all plant ns‐LTPs.
Non‐viral neuronal gene delivery mediated by the HC fragment of tetanus toxin
Authors: Knight Andrew , Carvajal Jaime , Schneider Holm , Coutelle Charles , Chamberlain Sue , Fairweather Neil
Published: March 2003

Many inherited neurological diseases and cancers could potentially benefit from efficient targeted gene delivery to neurons of the central nervous system. The nontoxic fragment C (HC) of tetanus toxin retains the specific nerve cell binding and transport properties of tetanus holotoxin. The HC fragment has previously been used to promote the uptake of attached proteins such as horseradish peroxidase, β‐galactosidase and superoxide dismutase into neuronal cells in vitro and in vivo. We report the use of purified recombinant HC fragment produced in yeast and covalently bound to polylysine [poly(K)] to enable binding of DNA. We demonstrate that when used to transfect cells, this construct results in nonviral gene delivery and marker gene expression in vitro in N18 RE 105 cells (a neuroblastoma × glioma mouse/rat hybrid cell line) and F98 (a glioma cell line). Transfection was dependent on HC and was neuronal cell type specific. HC may prove a useful targeting ligand for future neuronal gene therapy.
Purification, characterization and gene cloning of 6‐hydroxynicotinate  3‐monooxygenase from Pseudomonas fluorescens TN5
Authors: Nakano Hideo , Wieser Marco , Hurh Byungserk , Kawai Takahiro , Yoshida Toyokazu , Yamane Tsuneo , Nagasawa Toru
Published: March 2003

6‐Hydroxynicotinate 3‐monooxygenase, a membrane‐bound, 42‐kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6‐hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 m of FAD per 1 m of enzyme. The isolated enzyme was used for the synthesis of 2,5‐dihydroxypyridine, a precursor for the chemical synthesis of 5‐aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8‐kbp DNA fragment, which contains the ORF encoding 6‐hydroxynicotinic acid 3‐monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD‐binding site and is homologous to similar flavoproteins such as salicylate 1‐monoxygenase.
Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase
Authors: Vargiu Cristina , Cabella Claudia , Belliardo Sabina , Cravanzola Carlo , Grillo Maria Angelica , Colombatto Sebastiano
Published: March 2003

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1‐acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S‐adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.
Genetic and biochemical characterization of phosphofructokinase from the opportunistic pathogenic yeast Candida albicans
Authors: Lorberg Anja , Kirchrath Lutz , Ernst Joachim F. , Heinisch Jürgen J.
Published: March 2003

We have used the two PFK genes of Saccharomyces cerevisiae encoding the α and β‐subunit of the enzyme phosphofructokinase (Pfk) as heterologous probes to isolate fragments of the respective genes from the dimorphic pathogenic fungus Candida albicans. The complete coding sequences were obtained by combining sequences of chromosomal fragments and fragments obtained by inverse polymerase chain reaction (PCR). The CaPFK1 and CaPFK2 comprise open reading frames of 2961 bp and 2838 bp, respectively, encoding Pfk subunits with deduced molecular masses of 109 kDa and 104 kDa. The genes presumably evolved by a duplication event from a prokaryotic type ancestor, followed by another duplication. Heterologous expression in S. cerevisiae revealed that each gene alone was able to complement the glucose‐negative phenotype of a pfk1 pfk2 double mutant. In vitro Pfk activity in S. cerevisiae was not only obtained after coexpression of both genes, but also in conjunction with the respective complementary subunits from S. cerevisiae. This indicates the formation of functional hetero‐oligomers consisting of C. albicans and S. cerevisiae Pfk subunits. In C. albicans, specific Pfk activity was shown to decrease twofold upon induction of hyphal growth. CaPfk cross‐reacts with a polyclonal antiserum raised against ScPfk and displays similar allosteric properties, i.e. inhibition by ATP and activation by AMP and fructose 2,6‐bisphosphate.
Protection of mice against a lethal influenza virus challenge after immunization with yeast‐derived secreted influenza virus hemagglutinin
Authors: Saelens Xavier , Vanlandschoot Peter , Martinet Wim , Maras Marleen , Neirynck Sabine , Contreras Roland , Fiers Walter , Jou Willy Min
Published: March 2003

The A/Victoria/3/75 (H3N2‐subtype ) hemagglutinin (HA) gene was engineered for expression in Pichia pastoris as a soluble secreted molecule. The HA cDNA lacking the C‐terminal transmembrane anchor‐coding sequence was fused to the Saccharomyces cerevisiaeα‐mating factor secretion signal and placed under control of the methanol‐inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Growth of transformants on methanol‐containing medium resulted in the secretion of recombinant non‐cleaved soluble hemagglutinin (HA0s). Remarkably, the pH of the induction medium had an important effect on the expression level, the highest level being obtained at pH 8.0. The gel filtration profile and the reactivity against a panel of different HA‐conformation specific monoclonal antibodies indicated that HA0s was monomeric. Analysis of the N‐linked glycans revealed a typical P. pastoris type of glycosylation, consisting of glycans with 10–12 glycosyl residues.

  Mice immunized with purified soluble hemagglutinin (HA0s) showed complete protection against a challenge with 10 LD50 of mouse‐adapted homologous virus (X47), whereas all control mice succumbed. Heterologous challenge with X31 virus [A/Aichi/2/68 (H3N2‐subtype)], resulted in significantly higher survival rates in the immunized group compared with the control group. These results, together with the safety, reliability and economic potential of P. pastoris, as well as the flexibility and fast adaptation of the expression system may allow development of an effective recombinant influenza vaccine.
Examining the efficiency of receptor/G‐protein coupling with a cleavable β2‐adrenoceptor–Gsα fusion protein
Authors: Seifert Roland , Wenzel‐Seifert Katharina , Gether Ulrik , Lam Van T. , Kobilka Brian K.
Published: March 2003

Reconstitution of high‐affinity agonist binding at the β2‐adrenoceptor (β2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G‐protein of adenylyl cyclase, Gsα, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem.255, 369–382]. In a fusion protein of the β2AR and Gsα (β2AR‐Gsα), which has only a 1 : 1 stoichiometry of receptor and G‐protein, high‐affinity agonist binding and agonist‐stimulated GTP hydrolysis, guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G‐protein interaction, we constructed a fusion protein with a thrombin‐cleavage site between β2AR and Gsα (β2AR‐TS‐Gsα). β2AR‐TS‐Gsα efficiently reconstituted high‐affinity agonist binding, agonist‐stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves ≈ 70% of β2AR‐TS‐Gsα molecules in Sf9 membranes. Thrombin cleavage did not impair high‐affinity agonist binding and GTP[S] binding but strongly reduced ligand‐regulated GTPase activity and AC activity. We conclude that fusion of the β2AR to Gsα promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G‐protein. Dilution of Gsα in the membrane and release of activated Gsα into the cytosol can both prevent cleaved β2AR‐TS‐Gsα from undergoing multiple activation/deactivation cycles.
Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59fyn, GRB2 and Shc
Authors: Wellbrock Claudia , Schartl Manfred
Published: March 2003

Melanoma formation in Xiphoporus is initiated by overexpression of the EGFR‐related receptor tyrosine kinase Xmrk (Xiphoporus melanoma receptor kinase). This receptor is activated in fish melanoma as well as in a melanoma‐derived cell line (PSM) resulting in constitutive Xmrk‐mediated mitogenic signaling. In order to define the underlying signaling pathway(s), triggered by the activated Xmrk receptor, we attempted to identify its physiological substrates. Examination of the Xmrk carboxyterminus for putative tyrosine autophosphorylation sites revealed the presence of potential binding motifs for GRB2 as well as for Shc. Binding of these adaptor proteins to the Xmrk receptor was detected in vitro and in cells expressing the mrk kinase. The GRB2 and Shc interactions with the receptor could be disrupted individually by phosphotyrosine peptides containing putative Xmrk autophosphorylation sites, indicating direct binding of both proteins. Recruitment of GRB2 by the constitutively activated Xmrk receptor led to strong MAP kinase activation in Xiphoporus melanoma cells. We also identified a high‐affinity binding site for src‐kinases (pYEDL) in the Xmrk carboxyterminus. Competition experiments with phosphopeptides comprising this site confirmed that it is used for high‐affinity binding of Xiphoporus fyn (Xfyn) to Xmrk in melanoma cells. Thus, Xmrk can initiate different signaling pathways by using multiple substrate‐binding sites to trigger proliferation of pigment cells.
Primary structure and biochemical characterization of yeast  GTPase‐activating proteins with substrate preference for the transport GTPase Ypt7p
Authors: Vollmer Petra , Will Elke , Scheglmann Dietrich , Strom Molly , Gallwitz Dieter
Published: March 2003

Small GTPases of the Ypt/Rab family are regulators of vesicular protein trafficking in exo‐and endocytosis. GTPase‐activating proteins (GAP) play an important role as down regulators of GTPases. We here report the molecular cloning of a novel GAP‐encoding gene (GYP7, for GAP for Ypt7) by high expression from a Saccharomyces cerevisiae genomic library. The GYP7 gene encodes a hydrophilic protein with a molecular mass of 87 kDa. Comparison of its primary sequence with that of the three other known GAPs for transport GTPases, the yeast Gyp6 and Gyp1 proteins and the Rab3A‐GAP from rat brain, shows similarity between the yeast GAPs only. Like GYP6 and GYP1, GYP7 is not essential for yeast cell viability. Gyp7p was able to most effectively accelerate the intrinsic GTPase activity of Ypt7p. It was also active, but to a lesser extent, on Ypt31p, Ypt32p and Ypt1p. Ypt6p, Sec4p and the human H‐Ras protein did not serve as substrates. We also report the identification and cloning of a gene from the dimorphic yeast Yarrowia lipolytica that encodes a protein whose primary structure and biochemical activity are significantly related to those of Gyp7p from baker’s yeast.
Low‐temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature
Authors: Pellegrini Mariagiuseppina , Giardina Bruno , Castagnola Massimo , Olianas Alessandra , Sanna Maria Teresa , Fais Antonella , Messana Irene , Corda Marcella
Published: March 2003

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen‐binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2,3‐bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.
Folding stability of the kinetoplastid membrane protein‐11 (KMP‐11) from Leishmania infantum
Authors: Fuertes Miguel Angel , Berberich Christof , Lozano Rosa María , Gimenez‐Gallego Guillermo , Alonso Carlos
Published: March 2003

Kinetoplastid membrane protein‐11 (KMP‐11) is a major component of the cell surface of kinetoplastids, and acts as a potent B‐ and T‐cell immunogen during Leishmania infection. Here we report that the Leishmania infantum KMP‐11 secondary structure adopts mainly an α‐helical conformation at pH 7.5 and that its urea‐ and thermally‐induced unfolding constitute a fully reversible two‐step process. This allows estimation of a half‐denaturation temperature of ≈65 °C, a ΔGDH2O at 20 °C of ≈14.63 kJ·mol−1, and an increment of the reaction heat of ≈183.92 kJ·mol−1 and an entropy of ≈543.4 J·mol−1·deg−1, respectively, for the native‐denatured equilibrium of the KMP‐11 in solution. We also report that the KPM‐11 protein is induced to adopt a molten globule state at a pH range between pH 4 and pH 6. As a whole, the stability and the specific features of the denaturing effect induced by changes in pH are similar in KMP‐11 to various other lipoproteins.
Structural characterization of l‐aspartate oxidase and identification of an interdomain loop by limited proteolysis
Authors: Tedeschi Gabriella , Negri Armando , Ceciliani Fabrizio , Mattevi Andrea , Ronchi Severino
Published: March 2003

l‐Aspartate oxidase is the first enzyme in the de novo biosynthesis of pyridinic coenzymes in facultative aerobic organisms. The enzyme is FAD dependent and it shares common features with both the oxidase and the fumarate reductase classes of flavoproteins. In this report we focused our attention on the supersecondary structure of the molecule by means of limited proteolysis studies. Moreover the polymerization state of the protein at different pH and the interactions with NAD and its analogues are described. The results suggest that l‐aspartate oxidase is a monomer at pH values lower than 4.5 and a dimer at pH values higher than 6.5. The protein is organized in two major domains connected by a flexible loop located in the 120–140 region. The data obtained by limited proteolysis of the holo and the apo form in the presence and in the absence of substrates (fumarate and menadione), inhibitors (succinate) and NAD allows the proposition that both domains are involved in the binding of the flavin coenzyme. Moreover the data reported in this manuscript suggest that NAD inhibits l‐aspartate oxidase activity by competing with the flavin for the binding to the enzyme.
A unique primary structure, cDNA cloning and function of a galactose‐specific lectin from ascidian plasma
Authors: Abe Yukichi , Tokuda Miwako , Ishimoto Rika , Azumi Kaoru , Yokosawa Hideyoshi
Published: March 2003

The complete amino acid sequence of a galactose‐specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S‐pyridylethylated lectin. Peptide fragments were separated by reverse‐phase HPLC. The N‐terminal and C‐terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single‐chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross‐linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice‐repeated sequence, a fibrinogen‐related sequence and a C‐type lectin‐homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.
Post‐translational modification of nisin
Authors: Karakas Sen Asuman , Narbad Arjan , Horn Nikki , Dodd Helen M. , Parr Adrian J. , Colquhoun Ian , Gasson Michael J.
Published: March 2003

The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. As with all lantibiotics, nisin contains a number of dehydro‐residues and thioether amino acids that introduce five lanthionine rings into the target peptide. These atypical amino acids are introduced by post‐translational modification of a ribosomally synthesized precursor peptide. In certain cases, the serine residue, at position 33 of nisin, does not undergo dehydration to Dha33. With native nisin this partially processed form represents about 10% of the total peptide, whereas with the engineered variants, [Trp30]nisin A and [Lys27,Lys31]nisin A, the proportion of peptide that escapes full processing was found to be to approximately 50%. This feature of nisin biosynthesis was exploited in an investigation of the role of the NisB protein in pre‐nisin maturation. Manipulation of the level of NisB was achieved by cloning and overexpressing the plasmid‐encoded nisB gene in a range of different nisin‐producing strains. The resulting fourfold increase in the level of NisB significantly increased the efficiency of the dehydration reaction at Ser33. The final secreted product of biosynthesis by these strains was the homogenous form of the fully processed nisin (or nisin variant) molecule. The results presented represent the first experimental evidence for the direct involvement of the NisB protein in the maturation process of nisin.
Hydroxylation reaction catalyzed by the Burkholderia cepacia AC1100 bacterial strain
Authors: Martin Gaëlle , Dijols Sylvie , Capeillere‐Blandin Chantal , Artaud Isabelle
Published: March 2003

The Burkholderia cepacia AC1100 strain, known to degrade the herbicide, 2,4,5‐Trichlorophenoxyacetic acid (2,4,5‐T), is able to metabolize 4‐hydroxyarylaldehyde, not only into the corresponding acid, but also into a new hydroquinone, 2,5‐dihydroxyarylaldehyde. When incubated with resting AC1100 cells or cell‐free extracts, syringaldehyde and 3,5‐dimethoxy‐4‐hydroxybenzaldehyde were converted into such metabolites, identified by comparison of their mass and 1H‐NMR spectra with those of authentic chemically synthesized samples. With 5‐bromovanillin, only one metabolite was formed, the structure of which was identified as 2,5‐dihydroxy‐4‐methoxy‐6‐bromobenzaldehyde through 1H‐NMR two‐dimensional NOESY experiments. All these products result formally from a para hydroxylation of the phenol followed by the cis migration of the aldehyde. This reaction is the only one to be associated with the 2,4,5‐T degradation pathway, as the acid formation was retained when the AC1100 strain had lost its degradation ability. Through competitive experiments with halophenols and methimazole, an alternative substrate of flavin monooxygenase, the chlorophenol‐4‐monooxygenase was recognized to be the enzyme involved in the hydroxylation of 4‐hydroxyarylaldehyde. The purified enzyme, previously reported to catalyze the para hydroxylation or dehalogenating hydroxylation of chlorophenols, also promotes this hydroxylation reaction in the presence of NADH and FAD. The kcat value determined for the best substrate, syringaldehyde, 0.08 s−1, was about 20% of that obtained for 2,6‐dichlorophenol hydroxylation (0.38 s−1).
Microtubule motors in spindle and chromosome motility
Authors: Endow Sharyn A.
Published: March 2003

Many of the kinesin microtubule motor proteins discovered during the past 8–9 years have roles in spindle assembly and function or chromosome movement during meiosis or mitosis. The discovery of kinesin motor proteins with a clear involvement in spindle and chromosome motility, together with recent evidence that cytoplasmic dynein plays a role in chromosome distribution, has attracted great interest. The identification of microtubule motors that function in chromosome distribution represents a major advance in understanding the forces that underlie chromosome and spindle movements during cell division.
Activation of mitogen‐activated protein kinases (Erk1 and Erk2) cascade results in phosphorylation of NF‐M tail domains in transfected NIH 3T3 cells
Authors: Li Bing‐Sheng , Veeranna  , Jianguo Gu  , Grant P. , Pant Harish C.
Published: March 2003

Neurofilaments (NFs) are neuron‐specific intermediate filaments, and are the major cytoskeletal component in large myelinated axons. Lysine‐serine‐proline (KSP) repeats in the tail domains of high molecular weight NF proteins (NF‐M and NF‐H) are extensively phosphorylated in vivo in the axon. This phosphorylation in the tail domain has been postulated to play an important role in mediating neuron‐specific properties, including axonal caliber and conduction velocity. Recent studies have shown that the mitogen‐activated protein kinases (extracellular signal‐regulated kinases, Erk1 and Erk2) phosphorylate KSP motifs in peptide substrates derived from the NF‐M and NF‐H tail domains in vitro. However, it is not clear whether activation of the mitogen activated protein (MAP) kinase pathway is able to phosphorylate these domains in vivo. To answer this question, a constitutively active form of mitogen‐activated Erk activating kinase (MEK1) was cotransfected with an NF‐M expression construct into NIH 3T3 cells. The activated mutant, but not the dominant negative mutant, induced phosphorylation of NF‐M. In addition, it was shown that epidermal growth factor, which induces the MAP kinase cascade in NIH 3T3 cells, also activated endogenous Erk1 and Erk2 and NF‐M tail domain phosphorylation in the transfected cells. These results present direct evidence that in‐vivo activation of Erk1 and Erk 2 is sufficient for NF‐M tail domain phosphorylation in transfected cells.
Inhibition of an ecto‐ATP‐diphosphohydrolase by azide
Authors: Knowles Aileen F. , Nagy Agnes K.
Published: March 2003

Cell surface ATPases (ecto‐ATPases or E‐ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E‐ATPases have been divided into two subfamilies, ecto‐ATPases and ecto‐ATP‐diphosphohydrolases (ecto‐ATPDases). In the presence of either Mg2+ or Ca2+, ecto‐ATPDases, including proteins closely related to CD39, hydrolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto‐ATPases appear to lack these two properties. This report presents the first systematic kinetic study of a purified ecto‐ATPDase, the chicken oviduct ecto‐ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem.271, 16323–16331), with respect to ATP and ADP, and azide inhibition. Km values for ATP obtained at pH 6.4 and 7.4 are 10–30 times lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate, divalent ion, and pH. Nearly complete inhibition by 5 mm azide is obtained when MgADP is the substrate and when assays are conducted at pH 6–6.4. Azide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both Vmax and Km for ADP, it does not alter the Km for ATP. These results suggest that the apparent affinity of azide for the E·ADP complex is significantly greater than that for the free enzyme or E·ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.
Biochemical characteristics of Eiseniapore, a pore‐forming protein in the coelomic fluid of earthworms
Authors: Lange Sven , Kauschke Ellen , Mohrig Werner , Cooper Edwin L
Published: March 2003

The cytolytic protein Eiseniapore (38 kDa) from coelomic fluid of the earthworm Eisenia fetida functionally requires sphingomyelin as revealed by using mammalian erythrocytes and phospholipid vesicles. The effects of ions, glycoproteins and phospholipids were investigated for the two‐step Eiseniapore action mode, binding and pore formation in different assays. Eiseniapore lysis is activated by thiol groups but inhibited by metal ions. Eiseniapore binding to target membranes is inhibited by Eiseniapore‐regulating factor, vitronectin, heparin and lysophosphatidylcholine. Ca2+ and Mg2+ were found to be not necessary for membrane binding or lytic activity. Sphingomyelin was essential for Eiseniapore‐induced leakage of liposomes. We describe a cytolytic protein/toxin in Eiseniapore which differs from the established classification; it can be activated by thiol groups and is inhibited by sphingomyelin. Electron microscopy of erythrocyte membranes confirmed ring‐shaped structures (pores) with a central channel with outer (10 nm) and inner (3 nm) diameters as shown previously [Lange, S., Nüßler, F., Kauschke, E., Lutsch, G., Cooper, E.L. & Herrmann, A. (1997) J. Biol. Chem.272, 20 884–20 892] using artificial membranes. Functional evidence of pore formation by Eiseniapore was revealed as protection of lysis by carbohydrates occurred at an effective diameter above 3 nm. From these results, we suggest a plausible explanation for the mechanism by which components of the earthworm’s immune system destroy non‐self components.
Changes in the proton‐motive force in Escherichia coli in response to external oxidoreduction potential
Authors: Riondet Christophe , Cachon Rémy , Waché Yves , Alcaraz , Gérard , Diviès Charles
Published: March 2003

The pH homeostasis and proton‐motive force (Δp) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP). Only the internal pH value and, thus, the membrane pH gradient (ΔpH) component of the Δp is modified, while the membrane potential (ΔΨ) does not change in a significant way. Under reducing conditions (Eh < 50 mV at pH 7.0), E. coli decreases its Δp especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850‐mV ORP decrease). Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity. We propose that low ORP values de‐energize E. coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons.
Lys35 of PsaC is required for the efficient photoreduction of flavodoxin by photosystem I from Chlamydomonas reinhardtii
Authors: Meimberg Karen , Fischer Nicolas , Rochaix Jean‐David , Mühlenhoff Ulrich
Published: March 2003

The photoreduction of the oxidized and the semiquinone form of flavodoxin from Synechocystis sp. PCC 6803 by the photosystem I (PSI) of wild‐type Chlamydomonas reinhardtii and the mutant strains Lys35Asp, Lys35Glu and Lys35Arg was analysed by flash‐absorption spectroscopy to investigate the role of residue Lys35 of the PSI subunit PsaC in flavodoxin reduction. For PSI preparations from C. reinhardtii the reduction of oxidized flavodoxin was monoexponential and approached limiting electron transfer rates similar to those of cyanobacterial PSI from the wild‐type and the Lys35Arg mutant. For PSI from the Lys35Glu mutant, however, a ≈ 2.5‐fold smaller value was determined. The photoreduction of flavodoxin semiquinone by PSI from C. reinhardtii lacked fast first‐order kinetic components and, in contrast with PSI from cyanobacteria, displayed only a single concentration‐dependent phase. From this phase, second‐order rate constants were calculated for wild‐type PSI and PSI from the Lys35Arg mutant which were comparable to those of PSI from cyanobacteria. For PSI from the Lys35Glu and the Lys35Asp mutants the derived second‐order rate constants were 19 and 10 times smaller. Thus, the inversion of charge at position 35 of PsaC negatively affects the rate of electron transfer to both forms of flavodoxin, whereas PSI complexes that retain a positive charge at this position show wild‐type kinetics. However, the positive charge at this position of PsaC is not essential for flavodoxin photoreduction as the number of flavodoxin molecules reduced per PSI was similar for all of the PSI complexes investigated. In addition, chemical cross‐linking assays showed that the binary cross‐linking product between flavodoxin and PsaC of PSI from wild‐type C. reinhardtii was not formed with PSI complexes from the Lys13Asp and Lys35Glu mutants. This indicates that Lys35 of PsaC is probably essential for the chemical cross‐link between PsaC and flavodoxin. Taken together, these experiments show that Lys35 of PsaC plays a strikingly similar role in the electron transfer from PSI to both ferredoxin and flavodoxin.
Terminal truncations in Amp C β‐lactamase from a clinical isolate of Pseudomonas aeruginosa
Authors: Walther‐Rasmussen Jan , Johnsen Anders H. , Høiby Niels
Published: March 2003

AmpC β‐lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye‐affinity, size‐exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated β‐lactamase appeared to be both size‐ and charge‐homogeneous. The specific activities of the variants were very similar. MS of each isolated β‐lactamase form showed minor differences in molecular mass (range 40.0–40.8 kDa). MS of the β‐lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N‐terminal sequences of three of the β‐lactamases were identical to the published sequence [Lodge, J.M., Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J.272, 627–631], while two variants were truncated by two amino‐acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the β‐lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C‐terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five β‐lactamase isoforms are products of a single structural gene. The combinations of the identified N‐ and/or C‐terminal truncations explained the multiple pI values of the β‐lactamase isoforms.
The effect of α‐tocopherol on the thermotropic phase behaviour of dipalmitoylphosphatidylethanolamine
Authors: Wang Xiaoyuan , Quinn Peter J.
Published: March 2003

The effect of α‐tocopherol on the thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylethanolamine in mixtures containing 0, 2.5, 5, 10 and 20 mol% α‐tocopherol was examined using synchrotron X‐ray diffraction methods. Dispersions were equilibrated for at least 12 h at 4 °C before measurement. The phospholipid alone undergoes a lamellar gel to liquid‐crystalline phase transition at 66 °C during the initial heating scan. When codispersed with α‐tocopherol there was evidence of phase separation of α‐tocopherol‐rich domains from bilayers of pure phospholipid. The α‐tocopherol‐rich domains were identified from the relationship between scattering intensity of the phase and the proportion of α‐tocopherol in the mixture. In initial heating scans α‐tocopherol‐rich domains were characterized by broad lamellar repeat spacings (5.0 nm at 55 °C) in the small‐angle scattering region which first appears at ≈ 40 °C and increases in intensity with increasing temperature. In the presence of more than 5 mol% α‐tocopherol the wide‐angle scattering region showed two scattering profiles consisting of multiple peaks typical of lamellar crystal phases. The low‐temperature crystal phase, designated Lc1, was transformed into Lc2 phase at ≈ 50 °C. On further heating the Lc2 phase was transformed directly into inverted hexagonal phase at ≈56 °C and coexisted with the lamellar gel phase of the pure phospholipid. The inverted hexagonal phase coexists with the lamellar phase of the pure phospholipid, after transformation into lamellar liquid‐crystal phase at 66 °C, at least up to 70 °C. In cooling scans the d‐spacing of the inverted hexagonal phase decreases progressively and the scattering intensity weakens on cooling below the liquid‐crystal to gel phase transition temperature of the pure phospholipid. There is no evidence of formation of lamellar crystal phases during cooling so that the α‐tocopherol either mixes with the lamellar gel phase of the phospholipid without change in the structural parameters of the pure phospholipid or is completely phase separated from the phospholipid. The stoichiometry of phospholipid/α‐tocopherol in the α‐tocopherol‐rich lamellar crystal and inverted hexagonal phases is estimated to be ≈ 4 : 1. The structural changes observed in the study are related to the calorimetric transitions reported in independent work on these dispersions.