bluuebunny/crossref_metadata_2025 huggingface dataset:
Microperoxidase/H2O2‐Catalyzed Aromatic Hydroxylation Proceeds by a Cytochrome‐P‐450‐Type Oxygen‐Transfer Reaction Mechanism
Authors: Osman Ahmed M. , Koerts Janneke , Boersma Marelle G. , Boeren Sjef , Veeger Cees , Rietjens Ivonne M. C. M.
Published: July 2004

The mechanism of aromatic hydroxylation of aniline and phenol derivatives in a H2O2‐driven microperoxidase‐8(MP8)‐catalyzed reaction was investigated. It was shown that the reaction was not inhibited by the addition of scavengers of superoxide anion or hydroxyl radicals, which demonstrates that the reaction mechanism differs from that of the aromatic hydroxylation catalyzed by a horseradish peroxidase/dihydroxyfumarate system.

Additional experiments with 18O‐labelled H218O2 demonstrated that the oxygen incoiporated into aniline to give 4‐aminophenol originates from H2O2. Furthermore, it was found that the addition of ascorbic acid efficiently blocks all peroxidase‐type reactions that can be catalyzed by the MP8/H2O2 system, but does not inhibit the aromatic hydroxylation of aniline and phenol derivatives. Together, these observations exclude reaction mechanisms for the aromatic hydroxylation that proceed through peroxidase‐type mechanisms in which the oxygen incorporated into the substrate originates from O2 or H2O. The mechanism instead seems to proceed by an initial attack of the high‐valent iron‐oxo intermediate of MP8 on the π‐electrons of the aromatic ring of the substrate leading to product formation by a cytochrome‐P‐450‐type of σ‐O‐addition or oxygen‐rebound mechanism. This implies that MP8, which has a histidyl and not a cysteinate fifth axial ligand, is able to react by a cytochrome‐P‐450‐like oxygen‐transfer reaction mechanism.
On the Mechanism of Selenium Tolerance in Selenium‐Accumulating Plants
Authors: Neuhierl Bernhard , Böck August
Published: July 2004

Selected members of the genus Astragulus (Fabaceae) are known for their ability to accumulate high levels of selenium, mainly in the form of Se‐methyl‐selenocysteine. With the aid of cell cultures we have investigated the molecular basis for selenium tolerance of these plants. It is shown that cultured cells from a selenium‐accumulating Astragalus species synthesize Se‐methyl‐selenocysteine in contrast to those of a non‐accumulating species and do not unspecifically incorporate selenium into proteins. The purification and biochemical characterization of a selenocysteine methyltransferase from cultured Astragalus bisculatus cells is described, which does not accept cysteine as a substrate. We propose that this enzyme plays a crucial role in conferring selenium tolerance.
Key Role of Alkanoic Acids on the Spectral Properties, Activity, and Active‐Site Stability of Iron‐Containing Nitrile Hydratase from Brevibacterium R312
Authors: Kopf Marie‐Aude , Bonnet Didier , Artaud Isabella , Pétré Dominique , Mansuy Daniel
Published: July 2004

Interaction of n‐butyric acid with dialyzed nitrile hydratase from Brevibacterium R312, which is characterized by a charge‐transfer band at 680 nm and EPR signals typical of a low‐spin Fe(III) with Δg= 0.22, leads to a form displaying different spectral properties (λ= 710 nm, Δg= 0.31). Butyric acid also acts as a competitive inhibitor of nitrile‐hydratase‐catalyzed hydration of acrylonitrile with a Ki value of 0.9 mM. Formation of the complex between the enzyme and butyric acid is highly dependent on the concentration of the latter and on pH. When stored with high levels of butyric acid, nitrile hydratase is completely inactive. The active uncomplexed enzyme is restored under the high dilution conditions used for the enzymatic assays, while the complexed form is favored at acidic pH and is not formed at pH above 8. Furthermore, the inhibitory potency of butyric acid decreases upon increasing pH (IC50 increases from 0.8 mM at pH 6.2 to 12 mM at pH 8.2). These data show that nitrile hydratase interacts with the acid form of butyric acid with a high affinity (K′i≈ 4 μM at pH 7.2). At pH <3, the visible spectrum of the enzyme disappears, presumably because of demetallation, whereas that of the complex exhibits a charge‐transfer band shifted to 800 nm, the presence of butyric acid preventing nitrile hydratase from demetallation. Other linear carboxylic acids such as valeric and hexanoic acids behave similarly; they act as inhibitors of nitrile hydratase and protect the enzyme during storage. A structure of the nitrile hydratase active site interacting with butyric acid is tentatively proposed in which the latter is hydrogen‐bonded to the Fe(III)‐OH moiety. This interaction between butyric acid and nitrile hydratase should be considered when deducing the nature of nitrile hydratase active site and mechanism, from spectral and enzymatic data, since most results published previously have been obtained on nitrile hydratase containing large amounts of butyric acid and interpreted without taking into account the presence of this acid in the active site.
Structural Properties of the Putative Fusion Peptide of Hepatitis B Virus Upon Interaction with Phospholipids
Authors: Rodríguez‐crespo Ignacio , Gómez‐gutiérrez Julián , Encinar José A. , González‐ros José M. , Albar Juan P. , Peterson Darrell L. , Gavilanes Francisco
Published: July 2004

A peptide corresponding to the N‐terminal sequence of the S protein from hepatitis B virus (Met‐Glu‐Asn‐Ile‐Thr‐Ser‐Gly‐Phe‐Leu‐Gly‐Pro‐Leu‐Leu‐Val‐Leu‐Gln) has been previously shown to interact with phospholipids and promote vesicle aggregation, phospholipid mixing, and liposome leakage, as well as erythrocyte lysis [Rodríguez‐Crespo, I., Núñez, E., Gómez‐Gutiérrez, J., Yélamos, B., Albar, J. P., Peterson, D. L. & Gavilanes, F. (1995) J. Gen. Virol. 76, 301–308]. The conformation of this putative fusion peptide has been studied, both at low and high peptide concentrations, by means of circular dichroism and Fourier‐transform infrared spectroscopy, respectively. When the peptide is dissolved in trifluoroethanol, a significant population of α‐helical structure is found in spite of the proline residue at position 11. In contrast, this hydrophobic oligopeptide has a high tendency to form large β‐sheet aggregates in aqueous buffers. Most of these aggregates can be eliminated by centrifugation. The peptide remaining in the supernatant adopts a non‐ordered conformation. The aggregates can be dissociated by the anionic detergent sodium cholate, but the peptide still maintains an extended conformation. In the presence of acidic phospholipid vesicles, the putative fusion peptide adopts a highly stable β‐sheet conformation. Thus, unlike the fusion peptides of other viruses, an extended conformation seems to be the preferred structure when interacting with phospholipids. Such a conformation should be responsible for its membrane destabilization properties
The Structure of the O‐Specific Polysaccharide of Proteus penneri 52
Authors: Sidorczyk Zygmunt , Zych Krystyna , Świerzko Anna , Vinogradov Eugeny V. , Knirel Yuriy A.
Published: July 2004

An acidic O‐specific polysaccharide isolated from Proteus penneri 52 contains d‐galacturonic acid, 2‐acetamido‐2‐deoxy‐d‐glucose, and 2‐acetamido‐2‐deoxy‐d‐galactose in the ratio 1:2:2. On the basis of sugar analysis and NMR spectroscopy, which included one‐dimensional TOCSY, two‐dimensional COSY, heteronuclear 13C, 1H‐COSY, and rotating‐frame NOE spectroscopy, the following structure of the pentasaccharide repeating unit of the O‐specific polysaccharide was established:
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Cross‐reactivity of the anti‐P‐penneri 52 O‐serum with other strains of P. penneri isolated in Germany, Poland, USA, and Canada is discussed and a new Proteus serogroup is proposed.
The Basic Region/Helix‐Loop‐Helix/Leucine Repeat Transcription Factor USF Interferes with Ras Transformation
Authors: Aperlo Christel , Boulukos Kim E. , Pognonec Philippe
Published: July 2004

Upstream stimulatory factor (USF) is a transcription factor of the basic region/helix‐loop‐helix/leucine repeat family. It shares the same DNA‐binding sequence as the myc oncogene. Based on the three‐dimensional structures, its DNA‐binding domain is structurally related to that of Max, the partner of Myc. In addition, USF can form heterodimers with a related factor, Fos‐interacting protein/upstream stimulatory factor 2 (FIP/USF2), which has been shown to directly interact with Fos. In view of the provocative relationship of USF with other factors involved in cell proliferation, we investigated whether USF could also play a role in cellular growth control. In this study, we report that USF is not an oncogene, but interferes with Ras‐driven transformation. This inhibitory effect is independent of USF transactivating domains, but requires its DNA‐binding activity. However, the minimal USF DNA‐binding domain does not display this inhibitory effect, and even slightly enhances Ras transformation. On the basis of these data, we propose that USF may play an important role in the control of cell growth and proliferation, through both binding to promoter sequences and specific protein/protein interactions.
The Role of the Cytoplasmic Domain of α6 Integrin in the Assembly and Function of α6β1 and α6β4
Authors: De Melker Anneinieke A. , Sonnenberg Arnoud
Published: July 2004

We have studied the role of the cytoplasmic domain of α6 in the assembly and function of the α6β4 integrin, and compared it with the role of α6 in the assembly and function of α6β1, by transfection of cDNAs encoding cytoplasmic mutants of α6 into K562 cells with or without full‐length β4 cDNA. Des‐(1022–1050)‐α6, which contains a deletion C‐terminal to the GFFKR motif, was expressed in association with β1, but associated preferentially with β4, whereas the wild‐type α6 subunit associated efficiently with β1 and β4 Des‐(1016–1050)‐α6, which lacked also the GFFKR sequence, was only expressed at the cell surface when β4 was available. Transient expression in COS‐7 cells showed that des‐(1016–1050)‐α6 was retained in the endoplasmic reticulum as a monomer, which suggests that truncation of the cytoplasmic domain reduces the affinity of α6 for β1, particularly when the GFFKR sequence is absent. Although the GFFKR motif is not essential for association of α6 with β4, it increases the stability of the α6β4 integrin. The cytoplasmic domain of α6 is essential for inside‐out and outside‐in signaling via the α6β1 receptor, but not for adhesion via α6β4. We show that α6β4 is a constitutively active receptor. Thus, unlike adhesion by most other integrins, adhesion by α6β4 does not seem to depend on any active cellular process. Binding of α6β4 to ligand was only slightly affected by truncation of the α6 cytoplasmic domain N‐terminal to the GFFKR sequence and became partially dependent on metabolic energy. These data indicate that truncations of the cytoplasmic domain of the α6 subunit affect the assembly and function of α6β1 more strongly than those of α6β4. This difference may be due to the greater affinity of α6 for β4 than for β1, which makes α6β4 less susceptible to the effect of truncations.
Chimeras of Parvalbumin and Oncomodulin Involving Exchange of the Complete CD Site Show that the Ca2+/Mg2+ Specificity is an Intrinsic Property of the Site
Authors: Durussel Isabelle , Pauls Thomas L. , Cox Jos A. , Berchtold Martin W.
Published: July 2004

Rat parvalbumin (PV) and oncomodulin (OM) differ in the affinity and selectivity of metal binding to their CD site, which is a high‐affinity Ca2+/Mg2+‐mixed site in PV and a low‐affinity Ca2+‐specific site in OM. To assess to what degree the Ca2+/Mg2+ specificity and affinity of an EF‐hand motif in a protein is intrinsically determined by its sequence, the complete CD sites were exchanged, yielding two chimeras, [S41‐Q71]PV and [D41‐S71]OM. The optical characteristics of a Trp102, inserted in the hydrophobic core of PV, OM and the two chimeras, are very similar in all four proteins, which suggests that the hydrophobic core is qualitatively similar in the chimeras as in the parent proteins. Direct Ca2+ and Mg2+ binding monitored by flow dialysis and gel filtration revealed that [S41‐Q71]PV binds only one Mg2+ with an intrinsic affinity K′Mg2+ of 3.0×104 M−1 and two Ca2+ with an identical K′Ca2+ of 4.4×106 M−1, whereas [D41–S71]OM binds two Mg2+ with a mean K′Mg2+ of 2×104 M−1 and two Ca2+ with a K′Ca2+ of 1.3×104 M−1. K′Ca2+ of the CD site of [S41–Q71]PV was 2.5‐fold higher than of the CD site in [W102]OM, but 5–6‐fold lower than that of the CD site in [W102]PV. In [D41–S71]OM, K′Ca2+ of the CD site was twofold lower than in [W102]PV, but eightfold higher than in [W102]OM. These results indicate that the sequence of the CD site determines its Ca2+/Mg2+‐specificity, whereas its affinity for Ca2+ influenced by the protein into which the CD site is inserted. The inserted CD site in turn influences the affinity of the EF site to which it is paired in the host protein and the paired sites display an equalized affinity for Ca2+.

Mg2+ decreases the affinity of the chimeras for Ca2+, but not according to a simple competition model. The Mg2+ antagonism is much more pronounced in [D41–S71]OM than in [S41–Q71]PV, but in each chimera the CD and EF site are quantitatively affected in the same manner. Thus, [S41–Q71]PV which can only bind a single Mg2+ ion, displays a Ca2+/Mg2+‐antagonism for both sites with a K′Mg · compet of 2.3×102 M−1. These results confirm the ‘equalizer’ principle in the cation‐binding parameters of [S41–Q71]PV: both sites display the same Ca2+ affinity and Mg2+ antagonism. In [D41–S71]OM with its two Ca2+/Mg2+ sites the antagonism shows qualitatively the same complexity as in wild‐type PV, although it is somewhat weaker in amplitude.
Frustulins: Domain Conservation in a Protein Family Associated with Diatom Cell Walls
Authors: Kröger Nils , Bergsdorf Christian , Sumper Manfred
Published: July 2004

The outstanding feature of a diatom is the species‐specific design and ornamentation of the silica‐based cell wall, termed frustulum. A new frustulum is shaped in a specialized organelle (silica deposition vesicle) and secreted. Proteins in the lumen of this organelle may control the biomineralization process and are likely to remain associated with the mature cell wall. Therefore a study of the structures of proteins associated with the diatom cell wall was initiated. The complete primary structures of three cell wall proteins (denoted as frustulins) have been determined. In addition, partial amino acid sequences from two more cell wall components were obtained. From these data, a highly conserved domain has been identified as a common building block of diatom cell wall proteins that is repeated several time; per polypeptide chain together with polyproline/hydroxyproline or polyglycine spacers. All frustulins characterized so far, are synthesized as preproteins with a novel type of N‐terminal presequence.
Phosphorylation of Myeloid‐Related Proteins MRP‐14 and MRP‐8 During Human Neutrophil Activation
Authors: Guignard Florence , Mauel Jacques , Markert Michèle
Published: July 2004

The myeloid‐related proteins MRP‐14 and MRP‐8 and also p6, three calcium‐binding proteins of the S100 family, translocate to the membrane during human neutrophil activation with stimuli known to require extracellular calcium for activity. When phorbol 12‐myristate 13‐acetate (PMA, an extracellular calcium‐independent stimulus) is used, no translocation is observed. To characterize further the mechanisms involved in their translocation, phosphorylation of these proteins was studied.

Three isoforms of MRP‐14 were markedly phosphorylated in the membrane and in the cytosol upon activation with extracellular calcium‐dependent stimuli, such as opsonized zymosan, the calcium ionophore A23187, N ‐formylmethionylleucylphenylalanine in the presence of cytochalasin B and arachidonic acid, or upon extracellular calcium‐independent stimulation (PMA). In no case were p6 and a fourth, more basic isoform of MRP‐14, phosphorylated. In PMA‐activated cells, a phosphorylated acidic isoform of MRP‐8 was detected in the cytosol only. However, phosphorylated MRP‐8 represented only a small fraction of total MRP‐8. Cgp 41251, an inhibitor of protein kinase C (PKC), completely inhibited the phosphorylation of MRP‐8, and decreased cytosolic MRP‐14 phosphorylation. To test whether phosphorylated MRP‐8 could translocate, A23187, which induces translocation of the three S100 proteins, was added after PMA activation. This resulted in translocation of 18%±5% of phosphorylated MRP‐14 and 19%±1% of only nonphosphorylated MRP‐8. However, upon inhibition of PKC, translocation of MRP‐14 and MRP‐8 was increased up to 38%±7% and 34%±3% respectively. This suggests a putative role of phosphorylation and/or of PKC in the modulation of MRP‐14 and MRP‐8 translocation to the membrane.
Limited Proteolysis and Amino Acid Replacements in the Effector Region ofThermus thermophilusElongation Factor Tu
Authors: Zeidler Waltraud , Schirmer Norbert K. , Egle Christian , Ribeiro Sofia , Kreutzer Roland , Sprinzl Mathias
Published: July 2004

The effector region of the elongation factor Tu (EF‐Tu) fromThermus thermophiluswas modified by limited proteolysis or via site‐directed mutagenesis. The biochemical properties of the obtained EF‐Tu variants were investigated with respect to partial reactions of the functional cycle of EF‐Tu. EF‐Tu that was cleaved at the Arg59‐Gly60 peptide bond [EF‐Tu‐(1–59)/EF‐Tu‐(60–405)] bound GDP, EF‐Ts and aminoacyl‐tRNA, had normal intrinsic GTPase activity and was active in poly(U)‐dependent poly(Phe) synthesis. However, the GTPase activity of EF‐Tu‐(1–59)/EF‐Tu‐(60–405) was not stimulated byT. thermophilus70s ribosomes, and its GTP‐dissociation rate was increased compared with that of intact EF‐Tu. EF‐Tu cleaved at the Lys52‐Ala53 peptide bond has properties similar to EF‐Tu‐(1–59)/EF‐Tu‐(60–405). By means of site‐directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr inT thermophilusEF‐Tu. These amino acid substitutions did not substantially affect either the affinity of EF‐Tu · GTP for aminoacyl‐tRNA or the interactions with GDP, GTP or EF‐Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome‐induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome‐induced GTPase activity. The results demonstrate that the structure of the effector region of EF‐Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.
Methoxyamine‐Induced Mutagenesis of Nucleic Acids
Authors: Gdaniec Zofia , Ban Buu , Sowers Lawrence C. , Fazakerley G. Victor
Published: July 2004

We report the solution structure of two heptanucleotides each containing a central N4‐methoxycytosine, in one case paired with adenine on the opposite strand and the other with guanine. For the N4‐methoxycytosine · adenine pair, only the imino form of the N4‐methoxycytosine residue is observed and base pairing is in Watson‐Crick geometry. However, rotation of the methoxy group about the N‐OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4‐methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the cis conformation of the OCH3 group in the single strand, which inhibits base pair formation. For the N4‐methoxycytosine that is base paired with guanine, we observe an amino form in Watson‐Crick geometry in slow exchange with a base paired imino form in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 313 K. We have measured the exchange rate between the two forms at 303 K and observed a value of approximately 1s−1. The relative ratio of amino and imino forms of N4‐methoxycytosine is influenced by both the base that is in front and the temperature. Our results explain the preferential replacement of dTTP by N4‐methoxycytosine in primer elongation.
Clavin, a Type‐1 Ribosome‐Inactivating Protein from Aspergillus clavatus IF0 8605
Authors: Parente Dino , Raucci Giuseppe , Celano Bruna , Pacilli Aurelio , Zanoni Lorenzo , Canevari Silvana , Adobati Elena , Colnaghi Maria I. , Dosio Franco , Arpicco Silvia , Cattel Luigi , Mele Antonio , De Santis Rita
Published: July 2004

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the α‐sarcin family of ribosome‐inactivating proteins (RIPS). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17‐kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type‐1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell‐free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half‐life (t1/2β) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.
Coordinate Expression of Splice Variants of the Murine Pregnancy‐Specific Glycoprotein (PSG) Gene Family During Placental Development
Authors: Kromer Beril , Finkenzeller Daniela , Wessels Jennifer , Dveksler Gabriela , Thompson John , Zimmermann Wolfgang
Published: July 2004

The human and murine pregnancy‐specific glycoprotein (PSG) gene families encode a large number of closely related proteins which are abundantly expressed in the fetal trophoblast and secreted into the maternal circulation. Although the presence of a well conserved tripeptide sequence His or Arg‐Gly‐Asp or Glu or Lys (H/RGD/E/K) similar to the RGD motif found in extracellular matrix proteins hints towards a possible interaction with integrin‐type receptors, the function of this group of proteins related to the carcinoembryonic antigen family is still unknown. It is also not clear whether the various members of the PSG family exert the same function. Here we describe the cloning of two splice variants of Cea4 (Cea4a, Cea4b), a murine PSG family member, which lacks the RGD‐related consensus motif. Cea4a, like most of the other rodent PSG members, is composed of three immunoglobulin (Ig) variable‐like domains (N1–N3) and and one Ig constant‐like domain (A). In contrast, Cea4b lacks the N2 domain (N1N3A), demonstrating for the first time that PSG isoforms produced by alternative splicing also exist in mice. The mRNAs coding for Cea4a and Cea4b exhibit the same expression kinetics during placental development as found for two other murine PSGs, Cea2 and Cea3, which contain the RGD‐like motif. Expression starts after day 12.5 of embryonic development (E12.5) and maximum steady‐state levels are reached around E15.5–E17.5 as determined by RNase protection analyses. At E17.5, PSG transcripts can be detected exclusively in the spongiotrophoblast of the placenta. In addition, PCR analyses revealed that Cea2, Cea3, and Cea4 transcripts are also found in RNA from a pool of embryos (E12–E15) but are absent from a number of adult tissues tested (kidney, lung, testis, ovary, liver, brain, thymus, heart, spleen). These results indicate that the various PSG isoforms exert their function(s) at the same time during placental and embryonic development.
Bean Ribonuclease‐Like Pathogenesis‐Related Protein Genes (Ypr10) Display Complex Patterns of Developmental, Dark‐Induced and Exogenous‐Stimulus‐Dependent Expression
Authors: Walter Michael H. , Liu Jian‐Wei , Wünn Joachim , Hess Dieter
Published: July 2004

The intracellular pathogenesis‐related (PR) proteins of common bean (Phaseolus vulgaris L.) are encoded by a highly polymorphic family of at least 20 genes. One member, the Ypr10*c gene, has been isolated and characterised. The deduced amino acid sequence of the encoded protein, PR‐10, exhibits similarities to tree‐pollen allergens, to food allergens from celery and apple and to ginseng ribonuclease peptide sequences. We show by RNA blot analysis that the Ypr10 gene family, including Ypr10*c, is strongly expressed in bean roots. In leaves Ypr10 transcript levels are low in young and mature stages but are elevated during senescence and in diseased states. Dark treatment of leaves causes strong induction of Ypr10 transcripts, which is reversible by light, and diurnal rhythms of transcript accumulation during the night are observed. Ypr10 genes are responsive to external stimuli related to pathogen‐defence such as glutathione or salicylic acid. Transcriptional activity of a Ypr10*c promoter–β‐glucuronidase fusion gene in transgenic tobacco was observed in roots, in developing xylem and phloem of stems, and in the blade of senescent leaves, with highest levels at the onset of senescence. The most striking characteristic of developmental expression was the specific localisation of β‐glucuronidase activity in the transmitting tract of styles in flowers at anthesis. Feeding of various pathogen‐related and stress‐related stimuli to young tobacco leaves led to accumulation of GUS activity in leaf blades. We identify considerable spatio‐temporal similarities between reported expression patterns of Ypr10 genes and ribonuclease genes, which, together with the significant sequence similarity to the ginseng ribonuclease, support the hypothesis of a ribonuclease function for PR‐10 proteins and allow the prediction of possible biological roles.
A Low‐Molecular‐Mass GTP‐Binding Protein in the Cytosol of Germinated Wheat Embryos
Authors: Saccht Gian Attilio , Pirovano Livia , Lucchini Giorgio , Cocucci Sergio
Published: July 2004

A low‐molecular‐mass protein able to bind GTP in both native and SDS‐denaturating conditions was detected in the cytosol of embryos from wheat (Triticum aestivum L.) seeds germinated for 40 h. The protein fulfilled most of the distinguishing criteria common to eukaryotic small GTP‐binding proteins. It retained the ability to bind GTP after SDSPAGE and nitrocellulose blotting. The protein eluted from Sephadex G‐200 gel filtration with a Ve/Vo value corresponding to a molecular mass of 18 kDa, whereas on SDS/PAGE the molecular mass was 20 kDa. The native protein, which showed an intrinsic GTPase activity highly sensitive to NaF, bound the guanine nucleotide with high specificity and with a relatively high affinity (Kd, ? 85 nM). The GTP‐binding protein was not detectable in other subcellular fractions; in the microsomal fraction, two other peptides of low molecular mass (23.5 and 21.5 kDa) with GTP‐binding activity were detected. These results indicate that in the cytosolic fraction of germinating wheat embryos there is a 20–kDa protein which is biochemically similar to the known small GTP‐binding proteins that currently have been detected almost exclusively in the membrane fraction of plant material.
The C‐Terminal Domain of Light‐Harvesting Chlorophyll‐a/b‐Binding Protein is Involved in the Stabilisation of Trimeric Light‐Harvesting Complex
Authors: Kuttkat Andrea , Hartmann Annette , Hobe Stephan , Paulsen Harald
Published: July 2004

Light‐harvesting chlorophyll a/b‐binding protein (LHCP) can be reconstituted with pigments in detergent solution to yield stable monomeric light‐harvesting chlorophyll a/b complex (LHCII). This reconstitution is not significantly affected when up to ten amino acids are deleted on the C‐terminus of LHCP or when a tryptophan, which is 11 positions from the C terminus (W222), is exchanged with other amino acids [Paulsen, H. & Kuttkat, A. (1993) Photochem. Photobiol. 57, 139–142]. Here we show that the exchange of W222 with histidine or glycine completely abolishes the ability of the protein to assemble into trimeric LHCII, either upon reconstitution of monomeric complexes in detergent solution or upon insertion into isolated thylakoids. It is concluded that part of the hydrophilic domain on the C‐terminus of LHCP, although not essential for the formation of stable monomeric LHCII, is involved in trimer formation. The different degree to which various amino acids in place of W222 affect trainer formation suggests that a hydrophobic amino acid is needed in this position.
Class‐pi Glutathione S‐Transferase is Unable to Regain Its Native Conformation After Oxidative Inactivation by Hydrogen Peroxide
Authors: Sluis‐Cremer Nicolas , Naidoo Nirinjini , Dirr Heini
Published: July 2004

The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen‐peroxide‐mediated oxidation of class‐pi glutathione S‐transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non‐substrate‐ligand‐binding properties. The overall hydrodynamic volume of the oxidised class‐pi glutathione S‐transferase type‐1 homodimer (GSTP1‐1) is decreased, and its tertiary‐structural and secondary‐structural elements are changed with respect to the native protein. Structural differences appear to be prominent in domain 1 of oxidised GSTP1–1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidised protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1‐chloro‐2,4‐dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H‐site) for electrophilic substrates is negatively affected in that the Km and catalytic‐efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol‐treated oxidised GSTP1‐1 is able to regain its overall hydrodynamic volume; however, both its secondary‐structural and tertiary‐structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidised glutathione S‐transferase and dithiothreitol‐treated oxidised glutathione S‐transferase are less thermostable (tM= 55.5°C and 56.3°C, respectively) than the native enzyme (tM= 5°C). These results indicate that the class‐pi glutathione S‐transferase is unable to regain its native conformation after oxidative inactivation.
Rat Pristanoyl‐CoA Oxidase
Authors: Vanhooren Johannes C. T. , Fransen Marc , De Béthune Bernadette , Baumgart Eveline , Baes Myriam , Torrekens Sophie , Van Leuven Fred , Mannaerts Guy P. , Van Veldhoven Paul P.
Published: July 2004

The composite pristanoyl‐CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5′‐RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS‐gel electrophoresis.

The amino acid identity with rat palmitoyl‐CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl‐CoA oxidases (20%), suggesting that the palmitoyl‐CoA oxidase/pristanoyl‐CoA oxidase duplication occurred early in evolution.

The carboxy‐terminal tripeptide of pristanoyl‐CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal‐targeting signal‐1 import receptor indicated that SQL functions as a peroxisome‐targeting signal.

Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl‐CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl‐CoA oxidase gene, if present, is only poorly or not transcribed.
Lactoferrin: Similarity to Diamine Oxidase and Purification by Aminohexyl Affinity Chromatography
Authors: Houen Gunnar , Högdall Estrid V. , Barkholt Vibeke , Nørskov Leif
Published: July 2004

A protein reacting with a monoclonal antibody against human placental diamine oxidase was purified from the specific granules of human neutrofil granulocytes using affinity chromatography on aminohexyl‐divinylsulfonyl‐agarose. The protein had an Mr, determined by SDS/PAGE, corresponding to diamine oxidase, but had other properties which indicated that it might be a different protein. A combination of protein chemical techniques, including N‐terminal sequencing, identified the protein as lactoferrin, an iron‐containing protein with an Mr, of approximately 80000, a high isoelectric point and ferroxidase activity. Purified commercial lactoferrin was shown to bind to aminohexyl‐divinylsulfonyl‐agarose, and to be eluted in a heterogenous way from the matrix by amines and salt. Alignment of the sequences of diamine oxidase and lactoferrin showed that they are similar, indicating a common ancestry for these two different classes of metallo‐oxidases.
Characterisation of a Soluble Cytochrome c4 Isolated from Thiobacillus ferrooxidans
Authors: Cavazza Christine , Giudici‐Orticoni Marie‐Thérèse , Nitschke Wolfgang , Appia Corinne , Bonnefoy Violaine , Bruschi Mireille
Published: July 2004

A soluble c‐type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an α‐peak wavelength of 552 nm, a molecular mass of 21193 Da (as determined by mass spectroscopy), and a pI value of 9. N‐terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em= 385 mV±20 mV and Em= 480 mV±20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz= 3.1 and gz= 3.35. The gz= 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half‐times.

All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c‐type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.
Reexamination of Hormone‐Binding Properties of Protein Disulfide‐Isomerase
Authors: Guthapfel Régine , Gueguen Paul , Quemeneur Eric
Published: July 2004

Protein disulfide‐isomerase (PDI), an abundant multifunctional protein, has been described as a 3,3′,5‐triiodo‐l‐thyronine (T3)‐binding protein. As pointed out by several authors, the physiological significance of this hormone‐binding property has not been fully addressed. To clarify this point, we have analyzed the T3‐binding properties of purified PDI.

At equilibrium, T3 binds PDI at two binding sites: first, at a high‐affinity site with a Kd of 21 nM and a Bmax of 1.8×10−3, mol T3/mol PDI monomer, and second at a very low affinity site that is unsaturated up to 100 μM T3. Thus, T3 binding is mainly non‐specific and the specific part represents only about 0.2% of the protein monomer. Cross‐linking experiments at a concentration where mainly specific binding occurs indicate that PDI does not bind l‐T3 exclusively; a wide variety of analogs are also bound. Refolding of reduced denatured ribonuclease A by PDI is inhibited by T3 and analogs, and the inhibition profile reflects the binding properties very closely.

Since purified PDI displays neither the specificity expected for a physiological receptor, nor significant T3‐binding activity, results are discussed in terms of a necessary PDI association with another component to form a T3 receptor.
Identification of Protein‐Phosphatase‐1‐Binding Domains on the Glycogen and Myofibrillar Targetting Subunits
Authors: Johnson Deborah F. , Moorhead Greg , Caudwell F. Barry , Cohen Philip , Chen Yu Hua , Chen Mao Xiang , Cohen Patricia T. W.
Published: July 2004

The specificity of the catalytic subunit of protein phosphatase‐1 (PP1c) is modified by regulatory subunits that target it to particular subcellular locations. Here, we identify PP1C‐binding domains on GL and GM the subunits that target PPIC, to hepatic and muscle glycogen, respectively, and on M110 the subunit that targets PP1C to smooth muscle myosin. GM‐(G63–T93) interacted with PP1C and prevented GL from suppressing the dephosphorylation of glycogen phosphorylase, but it did not dissociate GL from PP1C or affect other characteristic properties of the PP1GL complex. These results indicate that GL contains two PP1C‐binding sites, the region which suppresses the dephosphorylation of glycogen phosphorylase being distinct from that which enhances the dephosphorylation of glycogen synthase. At higher concentrations, GM‐(G63–N75) had the same effect as GM‐(G63–T93), but not if Ser67 was phosphorylated by cyclic‐AMP‐dependent protein kinase. Thus, phosphorylation of Ser67 dissociates GM from PP1C because phosphate is inserted into the PP1C‐binding domain of GM M110‐(M1‐E309) and M110‐(M1‐F38), but not M110‐(D39–E309), mimicked the M110 subunit in stimulating dephosphorylation of the smooth muscle myosin P‐light chain and heavy meromyosin in vitro. However, in contrast to the M110 subunit and M110‐(M1‐E309), neither M110‐(M1‐F38) nor M1110‐(D39–E309) suppressed the PPC‐catalysed dephosphorylation of glycogen phosphorylase. These observations suggest that the region which stimulates the dephosphorylation of myosin is situated within the N‐terminal 38 residues of the M110 subunit, while the region which suppresses the dephosphorylation of glycogen phosphorylase requires the presence of at least part of the region 39–309 which contains seven ankyrin repeats. M110‐(M1‐F38) displaced GL from PPIC while GM‐(G63‐T93) displaced M110 from PP1Cin vitro. These observations indicate that the region(s) of PP1c that interact with GM/GL and M110 overlap, explaining why different forms of PP1C contain just a single targetting subunit.
Regions of the 110‐kDa Regulatory Subunit M110 Required for Regulation of Myosin‐Light‐Chain‐Phosphatase Activity in Smooth Muscle
Authors: Gailly Philippe , Wu Xuqiong , Haystead Timothy A. J. , Somlyo Andrew P. , Cohen Patricia T. W. , Cohen Philip , Somlyo Avril V.
Published: July 2004

To characterize the in situ interactions between the subunits (regulatory 110kDa, M110; 21‐kDa, M21 and catalytic, 37‐kDa, PP1C) of smooth muscle myosin phosphatase (SMPP‐1M), we determined, in Triton‐X‐100‐permeabilized rabbit portal vein contracted with microcystin‐LR, the ability of the following fragments of M110 to regulate relaxation induced by exogenous PP1C (a) M110 purified from pig bladder; (b) the 72.5‐kDa N‐terminal fragment expressed from rat kidney cDNA [glutathione‐S ‐transferase–M110(11–612)‐peptide]; (c) a 58‐kDa fragment, the N‐terminal degradation product of M110 (M58; (d) two fragments expressed from rat aorta cDNA [M110‐(1–309)‐peptide and M110‐(39–309)‐peptide]; a synthetic fragment of M110 [M110‐(1–38)‐peptide]. The M110/M21, complex accelerated approximately 1.6‐fold the rate of dephosphorylation of the myosin P‐light chain and also relaxation induced by PP1c The glutathione‐S ‐transferase–M110‐(11–612)‐peptide and the M58 fragments, as well as the M110(1–309)‐peptide and, at higher concentration, M110‐(1–38)‐peptide, had similar effects that did not require the M21, subunit. Arachidonic acid, known to dissociate PP1C. from the native holoenzyme and inhibit SMPP‐1M activity, inhibited the regulatory action of the M110/M21 complex on PP1C activity and, to a lesser extent that, of the glutathione‐S ‐transferase‐ M110‐(11–612)‐peptide, hut not that of the M58 fragment or of the shorter peptides. We conclude that, consistent with in vitro studies [8], the N‐terminal sequence (1–309) of the M110 subunit is also sufficient to enhance the activity of PP1C for myosin in muscle. However, its C‐terminal half (downstream from the M58 fragment) is required for inhibition by arachidonic acid. In contrast to the effect of the M110 subunit and its fragments, a peptide, corresponding to part of the PP1C‐binding site of the regulatory glycogen‐binding subunit from skeletal muscle GM [GM‐(63–93)‐peptide], specifically slowed the relaxation, induced by tlash photolysis of diazo‐2, of Triton X‐100‐permeabilized femoral artery strips, and inhibited the holoenzyme‐induced relaxation in the portal vein, suggesting that the GM subunit can compete with the regulatory effect of M110 on PP1C in smooth muscle.
Electron‐Dense Granules in Desulfovibrio gigas do not Consist of Inorganic Triphosphate but of a Glucose Pentakis(Diphosphate)
Authors: Hensgens Charles M. H. , Santos Helena , Zhang Chenghong , Kruizinga Wim H. , Hansen Theo A.
Published: July 2004

Under certain growth conditions the sulfate‐reducing bacterium Desulfovibrio gigas forms electron‐dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH+4 concentration and reinvestigated the nature of the electron‐dense bodies. Energy‐dispersive X‐ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P‐NMR spectra also lacked the peaks in the −22‐ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron‐dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron‐dense bodies of D. gigas contained a novel metabolite which was identified as α‐glucose 1,2,3,4,6‐pentakis(diphosphate).
Isolation and Characterization of a 14.5‐kDa Trichloroacetic‐Acid‐Soluble Translational Inhibitor Protein from Human Monocytes that is Upregulated Upon Cellular Differentiation
Authors: Schmiedeknecht Gerno , Kerkhoff Claus , Orsó Evelyn , Stöhr Josef , Aslanidis Charalampos , Nagy György M. , Knuechel Ruth , Schmitz Gerd
Published: July 2004

A trichloroacetic‐acid‐soluble 14.5‐kDa protein (p14.5) has been isolated from human mononuclear phagocytes (MNP) by a combination of trichloroacetic acid extraction, preparative electrophoresis and hydrophobic affinity chromatography; five tryptic peptides were subjected to protein sequencing. The full‐length cDNA of the protein was cloned and sequenced from a λgt11 human liver library. The cDNA showed a remarkable similarity to a rat protein preferentially expressed in hepatocytes and renal tubular epithelial cells. The encoded protein is 137 amino acids long and similar to members of a new hypothetical family of small proteins with presently unknown function, named YER057c/YJGF. Human recombinant p14.5 inhibits in vitro protein synthesis in a rabbit reticulocyte lysate system. Unlike other inhibitors of protein synthesis, p14.5 is not phosphorylated despite the presence of putative phosphorylation sites. The p14.5 mRNA is weakly expressed in freshly isolated monocytes but is significantly upregulated when these monocytes are subjected to differentiation. This is also reflected by a differentiation‐dependent increase in the protein concentration as demonstrated by immunoblots from cytosolic fractions and fluorescence‐activated flow cytometry of permeabilized cells. A differentiation‐dependent mRNA and protein expression of p14.5 is further suggested by the observation of a low expression in a variety of liver and kidney tumor cells and a high expression in fully differentiated cells as assessed by immunohistochemistry and northern blots. The highest mRNA expression was found in hepatocytes and renal distal tubular epithelial cells and only weak expression was found in other human tissues as evaluated by northern blot analysis. The preferential localization of the immunoreaction product seemed to be cytoplasmatic but, in less differentiated cells, nuclear labeling was occasionally visible. Immunoblotting of subcellular fractions confirmed these data. The high degree of evolutionary conservation of p14.5, the considerable upregulation during cellular differentiation and its potential role as a translational inhibitor may reflect an involvement in basic cellular mechanisms, e.g. a differentiation‐dependent regulation of protein synthesis in hepatocytes, renal tubular epithelial cells, smooth muscle cells and MNP.
Interaction of Amino Acid Residues at Positions 8–15 of Secretin with the N‐Terminal Domain of the Secretin Receptor
Authors: Gourlet Philippe , Vilardaga Jean‐Pierre , De Neef Philippe , Vandermeers André , Waelbroeck Magali , Bollen Alex , Robberecht Patrick
Published: July 2004

The ability of secretin, PACAP‐(1–27)‐peptide, and ten hybrid peptides to recognize and activate the rat secretin and vasoactive intestinal polypeptide (PACAP type II VIP1) receptors was tested on recombinant Chinese hamster ovary (CHO) cell lines. PACAP had a 2500‐fold lower affinity than secretin for the secretin receptor, and secretin had a 300‐fold lower affinity than PACAP for the VIP1 receptor. Amino acids 8, 13, and 15 of the PACAP molecule contributed significantly to the low affinity of PACAP for the secretin receptor. The amino acids at positions 5, 9, 10, 15, 16, and unidentified amino acid(s) between positions 17–20 made limited contributions to the low affinity of secretin for the VIP1 receptor.

To identify the receptor region that interacts with these amino acids, we constructed chimeric receptors, which consist either of the N‐terminal extracellular part of the secretin receptor and the core of the VIP1 receptor (N‐Sn/VIP1r) or the N‐terminal extracellular part of the VIP1 receptor and the core of the secretin receptor (N‐VIP1/Snr), and tested the ability of the hybrid ligands to activate the adenylate cyclase of CHO cells expressing these chimeric receptors.

The N‐Sn/VIP1 receptors had a higher affinity for secretin than for PACAP. The hybrid peptide 6 that consists of the PACAP‐(1–8)‐Sn‐(9–15)‐PACAP‐(16–27)‐peptide sequence had a 30‐fold to 200‐fold higher potency than either parent peptide for the chimeric receptor, which suggests that while the N‐ and/or C‐terminal part of the peptide interact with the transmembrane domain of the receptor, the discriminator region 9–15 recognizes the extracellular N‐terminal domain of the receptor. This was confirmed by the observation that, out of all the peptides tested, hybrid 6 had the weakest potency for activation of the N‐VIP1/Sn chimeric receptors.
Characterization of the Interaction of the Monomeric GTP‐Binding Protein Rab3a with Geranylgeranyl Transferase II
Authors: Johannes Ludger , Perez Franck , Laran‐Chich Marie‐Pierre , Henry Jean‐Pierre , Darchen François
Published: July 2004

The monomeric GTP‐binding protein Rab3a controls exocytosis in neuroendocrine and neuronal cells. Like other members of the Rab family, Rab3a is posttranslationally modified by the addition of hydrophobic geranylgeranyl groups to its C‐terminus. The geranylgeranylation reaction is catalysed by the heterotrimeric geranylgeranyl transferase II. We describe the cDNA cloning of the β‐subunit of human geranyl‐geranyl transferase II by means of the yeast two‐hybrid system. The human enzyme, which is 49% and 96% similar to yeast and rat isoforms, respectively, can complement the β‐subunit deficiency in the yeast strain ANY119. Furthermore, by means of the two‐hybrid system and in vitro geranylgeranylation reactions with purified recombinant rat geranylgeranyl transferase II, we have characterized Rab3a domains implicated in the interaction with geranylgeranyl transferase II. We find that the N‐terminus, the effector loop, the hypervariable region of the C‐terminus, and the geranylgeranyl‐acceptor cysteines have roles in this interaction. The GDP‐bound form of Rab3a is the preferred substrate of geranylgeranyl transferase II.