bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel meta‐cleavage dioxygenase that cleaves a carboxyl‐group‐substituted 2‐aminophenol
Authors: Takenaka Shinji , Asami Tokiko , Orii Chika , Murakami Shuichiro , Aoki Kenji
Published: March 2003

A bacterial strain that grew on 4‐amino‐3‐hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4‐amino‐3‐hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110‐fold increase in specific activity. The purified enzyme was characterized as a meta‐cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4‐amino‐3‐hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4‐amino‐3‐hydroxybenzoate 2,3‐dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21‐kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4‐amino‐3‐hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 µm and 12 µmol·min−1·(mg protein)−1, respectively. Of the 2‐aminophenols tested, only 4‐aminoresorcinol and 6‐amino‐m‐cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2‐aminophenol 1,6‐dioxygenase, in molecular mass, subunit structure and catalytic properties.
Structural analysis of deacylated lipopolysaccharide of Escherichia coli strains 2513 (R4 core‐type) and F653 (R3 core‐type)
Authors: Müller‐Loennies Sven , Lindner Buko , Brade Helmut
Published: March 2003

Lipopolysaccharide (LPS) of Escherichia coli strain 2513 (R4 core‐type) yielded after alkaline deacylation one major oligosaccharide by high‐performance anion‐exchange chromatography (HPAEC) which had a molecular mass of 2486.59 Da as determined by electrospray ionization mass spectrometry. This was in accordance with the calculated molecular mass of a tetraphosphorylated dodecasaccharide of the composition shown below. NMR‐analyses identified the chemical structure as

where l‐α‐d‐Hep is l‐glycero‐α‐d‐manno‐heptopyranose and Kdo is 3‐deoxy‐α‐d‐manno‐oct‐2‐ulopyranosylonic acid and all hexoses are present as d‐pyranoses.

We have also isolated the complete core‐oligosaccharides of E. coli F653 LPS for which only preliminary data were available and investigated the deacylated LPS by NMR and MS. The proposed structure determined previously by methylation analysis was confirmed and is shown below.

In addition we have quantified the side‐chain heptose substitution of the inner core with GlcpN (≈ 30%) and confirmed that this sugar is only present when the phosphate at the second l,d‐Hepp residue is absent.
Biophysical characterization of the interaction of high‐density lipoprotein (HDL) with endotoxins
Authors: Brandenburg Klaus , Jürgens Gudrun , Andrä Jörg , Lindner Buko , Koch Michel H. J. , Blume Alfred , Garidel Patrick
Published: March 2003

The interaction of bacterial endotoxins [lipopolysaccharide (LPS) and the ‘endotoxic principle’ lipid A], with high‐density lipoprotein (HDL) from serum was investigated with a variety of physical techniques and biological assays. HDL exhibited an increase in the gel to liquid crystalline phase transition temperature Tc and a rigidification of the acyl chains of the endotoxins as measured by Fourier‐transform infrared spectroscopy and differential scanning calorimetry. The functional groups of the endotoxins interacting with HDL are the phosphates and the diglucosamine backbone. The finding of phosphates as target groups is in accordance to measurements of the electrophoretic mobility showing that the zeta potential decreases from −50 to −60 mV to −20 mV at binding saturation. The importance of the sugar backbone as further target structure is in accordance with the remaining negative potential and competition experiments with polymyxin B (PMB) and phase transition data of the system PMB/dephosphorylated LPS. Furthermore, endotoxin binding to HDL influences the secondary structure of the latter manifesting in a change from a mixed α‐helical/β‐sheet structure to a predominantly α‐helical structure. The aggregate structure of the lipid A moiety of the endotoxins as determined by small‐angle X‐ray scattering shows a change of a unilamellar/inverted cubic into a multilamellar structure in the presence of HDL. Fluorescence resonance energy transfer data indicate an intercalation of pure HDL, and of [LPS]–[HDL] complexes into phospholipid liposomes. Furthermore, HDL may enhance the lipopolysaccharide‐binding protein‐induced intercalation of LPS into phospholipid liposomes. Parallel to these observations, the LPS‐induced cytokine production of human mononuclear cells and the reactivity in the Limulus test are strongly reduced by the addition of HDL. These data allow to develop a model of the [endotoxin]/[HDL] interaction.
Determinants of antagonist binding at the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor subunit, GluR‐D
Authors: Jouppila Annukka , Pentikäinen Olli T. , Settimo Luca , Nyrönen Tommi , Haapalahti Jukka‐Pekka , Lampinen Milla , Mottershead David G. , Johnson Mark S. , Keinänen Kari
Published: March 2003

Previous structural and mutagenesis studies indicate that the invariant α‐amino and α‐carboxyl groups of glutamate receptor agonists are engaged in polar interactions with oppositely charged, conserved arginine and glutamate residues in the ligand‐binding domain of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor. To examine the role of these residues (R507 and E727 in the GluR‐D subunit) in the discrimination between agonists and antagonists, we analyzed the ligand‐binding properties of homomeric GluR‐D and its soluble ligand‐binding domain with mutations at these positions. Filter‐binding assays using [3H]AMPA, an agonist, and [3H]Ro 48–8587, a high‐affinity antagonist, as radioligands revealed that even a conservative mutation at R507 (R507K) resulted in the complete loss of both agonist and antagonist binding. In contrast, a negative charge at position 727 was necessary for agonist binding, whereas the isosteric mutation, E727Q, abolished all agonist binding but retained high‐affinity binding for [3H]Ro 48–8587, displaceable by 7,8‐dinitroquinoxaline‐2,3‐dione. Competition binding studies with antagonists representing different structural classes in combination with ligand docking experiments suggest that the role of E727 is antagonist‐specific, ranging from no interaction to weak electrostatic interactions involving indirect and direct hydrogen bonding with the antagonist molecule. These results underline the importance of ion pair interaction with E727 for agonist activity and suggest that an interaction with R507, but not with E727, is essential for antagonist binding.
Novel fish hypothalamic neuropeptide
Authors: Sawada Kaori , Ukena Kazuyoshi , Satake Honoo , Iwakoshi Eiko , Minakata Hiroyuki , Tsutsui Kazuyoshi
Published: March 2003

Recently, we identified novel avian and amphibian hypothalamic neuropeptides that inhibited gonadotropin release and stimulated growth hormone release. They were characterized by a similar structure including the C‐terminal LPLRF‐NH2 motif. To clarify that the expression of these novel hypothalamic neuropeptides is a conserved property in vertebrates, we characterized a cDNA encoding a similar novel peptide, having LPLRF‐NH2 from the goldfish brain, by a combination of 3′ and 5′ rapid amplification of cDNA ends (RACE). The deduced peptide precursor consisted of 197 amino acid residues, encoding three putative peptide sequences that included ‐LPXRF (where X is L or Q) at their C‐termini. Mass spectrometric analyses revealed that a tridecapeptide (SGTGLSATLPQRF‐NH2) was derived from the precursor in the brain as an endogenous ligand. Southern blotting analysis of reverse‐transcriptase‐mediated PCR products demonstrated a specific expression of the goldfish peptide gene in the diencephalon. In situ hybridization revealed the cellular localization of goldfish peptide mRNA in the nucleus posterioris periventricularis in the hypothalamus. Immunoreactive cell bodies were also restricted to the the nucleus posterioris periventricularis and the nervus terminalis and immunoreactive fibers were distributed in several brain regions including the nucleus lateralis tuberis pars posterioris and pituitary. Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C‐terminal ‐LPQRF‐NH2 sequence, which may possess multiple regulatory functions and act, at least partly, on the pituitary to regulate pituitary hormone release.
Leishmania donovani methionine adenosyltransferase
Authors: Pérez‐Pertejo Yolanda , Reguera Rosa M. , Villa Hector , García‐Estrada Carlos , Balaña‐Fouce Rafael , Pajares Maria A. , Ordóñez David
Published: March 2003

Methionine adenosyltransferase (MAT, EC 2.5.1.6)‐mediated synthesis of S‐adenosylmethionine (AdoMet) is a two‐step process consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities, AdoMet synthesis and tripolyphosphate hydrolysis, can be measured separately is particularly useful when the site‐directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. The present report describes the cloning and subsequent functional refolding, using a bacterial expression system, of the MAT gene (GenBank accession number AF179714) from Leishmania donovani, the etiological agent of visceral leishmaniasis. The absolute need to include a sulfhydryl‐protection reagent in the refolding buffer for this protein, in conjunction with the rapid inactivation of the functionally refolded protein by N‐ethylmaleimide, suggests the presence of crucial cysteine residues in the primary structure of the MAT protein. The seven cysteines in L. donovani MAT were mutated to their isosterical amino acid, serine. The C22S, C44S, C92S and C305S mutants showed a drastic loss of AdoMet synthesis activity compared to the wild type, and the C33S and C47S mutants retained a mere 12% of wild‐type MAT activity. C106S mutant activity and kinetics remained unchanged with respect to the wild‐type. Cysteine substitutions also modified PPPi cleavage and AdoMet induction. The C22S, C44S and C305S mutants lacked in tripolyphosphatase activity altogether, whereas C33S, C47S and C92S retained low but detectable activity. The behavior of the C92S mutant was notable: its inability to synthesize AdoMet combined with its retention of tripolyphosphatase activity appear to be indicative of the specific involvement of the respective residue in the first step of the MAT reaction.
Proteomic identification of all plastid‐specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit
Authors: Yamaguchi Kenichi , Subramanian Alap R.
Published: March 2003

Six ribosomal proteins are specific to higher plant chloroplast ribosomes [Subramanian, A.R. (1993) Trends Biochem. Sci.18, 177–180]. Three of them have been fully characterized [Yamaguchi, K., von Knoblauch, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28455–28465; Yamaguchi, K. & Subramanian, A. R. (2000) J. Biol. Chem. 275, 28466–28482]. The remaining three plastid‐specific ribosomal proteins (PSRPs), all on the small subunit, have now been characterized (2D PAGE, HPLC, N‐terminal/internal peptide sequencing, electrospray ionization MS, cloning/ sequencing of precursor cDNAs). PSRP‐3 exists in two forms (α/β, N‐terminus free and blocked by post‐translational modification), whereas PSRP‐2 and PSRP‐4 appear, from MS data, to be unmodified. PSRP‐2 contains two RNA‐binding domains which occur in mRNA processing/stabilizing proteins (e.g. U1A snRNP, poly(A)‐binding proteins), suggesting a possible role for it in the recruiting of stored chloroplast mRNAs for active protein synthesis. PSRP‐3 is the higher plant orthologue of a hypothetical protein (ycf65 gene product), first reported in the chloroplast genome of a red alga. The ycf65 gene is absent from the chloroplast genomes of higher plants. Therefore, we suggest that Psrp‐3/ycf65, encoding an evolutionarily conserved chloroplast ribosomal protein, represents an example of organelle‐to‐nucleus gene transfer in chloroplast evolution. PSRP‐4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis. We would therefore suggest that PSRP‐4 is an example of gene capture (via horizontal gene transfer) during chloro‐ribosome emergence. Orthologues of all six PSRPs are identifiable in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database. All six PSRPs are nucleus‐encoded. The cytosolic precursors of PSRP‐2, PSRP‐3, and PSRP‐4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively. Functions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed.
Fluorescence and FTIR study of pressure‐induced structural modifications of horse liver alcohol dehydrogenase (HLADH)
Authors: Trovaslet Marie , Dallet‐Choisy Sandrine , Meersman Filip , Heremans Karel , Balny Claude , Legoy Marie Dominique
Published: March 2003

The process of pressure‐induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1–600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure‐induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native‐like state was assumed. Higher pressures (100–400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule‐like state was proposed. Further increase of pressure (400–600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure‐induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.
Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase
Authors: Leemhuis Hans , Dijkstra Bauke W. , Dijkhuizen Lubbert
Published: March 2003

Cyclodextrin glycosyltransferase (CGTase) uses an α‐retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg·L−1 culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one‐substrate reaction, whereas the other two transglycosylation reactions are two‐substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction).

Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme–substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.
Affinity purification‐mass spectrometry
Authors: Bauer Andreas , Kuster Bernhard
Published: March 2003

Multi‐protein complexes are emerging as important entities of biological activity inside cells that serve to create functional diversity by contextual combination of gene products and, at the same time, organize the large number of different proteins into functional units. Many a time, when studying protein complexes rather than individual proteins, the biological insight gained has been fundamental, particularly in cases in which proteins with no previous functional annotation could be placed into a functional context derived from their ‘molecular environment’. In this minireview, we summarize the current state of the art for the retrieval of multiprotein complexes by affinity purification and their analysis by mass spectrometry. The advances in technology made over the past few years now enable the study of protein complexes on a proteomic scale and it can be anticipated that the knowledge gathered from such projects will fuel drug target discovery and validation pipelines and that the technology is also going to prove valuable in the emerging field of systems biology.
Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC‐MS
Authors: Fischer Eliane , Sauer Uwe
Published: March 2003

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC‐MS‐derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC‐MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1‐13C] and [U‐13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner– Doudoroff pathway in wild‐type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6‐phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.
Acryloyl‐CoA reductase from Clostridium propionicum
Authors: Hetzel Marc , Brock Matthias , Selmer Thorsten , Pierik Antonio J. , Golding Bernard T. , Buckel Wolfgang
Published: March 2003

Acryloyl‐CoA reductase from Clostridium propionicum catalyses the irreversible NADH‐dependent formation of propionyl‐CoA from acryloyl‐CoA. Purification yielded a heterohexadecameric yellow–greenish enzyme complex [(α2βγ)4; molecular mass 600 ± 50 kDa] composed of a propionyl‐CoA dehydrogenase (α2, 2 × 40 kDa) and an electron‐transferring flavoprotein (ETF; β, 38 kDa; γ, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per α2βγ subcomplex (149 kDa) was determined. A substrate alternative to acryloyl‐CoA (Km = 2 ± 1 µm; kcat = 4.5 s−1 at 100 µm NADH) is 3‐buten‐2‐one (methyl vinyl ketone; Km = 1800 µm; kcat = 29 s−1 at 300 µm NADH). The enzyme complex exhibits acyl‐CoA dehydrogenase activity with propionyl‐CoA (Km = 50 µm; kcat = 2.0 s−1) or butyryl‐CoA (Km = 100 µm; kcat = 3.5 s−1) as electron donor and 200 µm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N‐terminus of the dimeric propionyl‐CoA dehydrogenase subunit is similar to those of butyryl‐CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N‐termini of the β and γ subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl‐CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans‐2‐enoyl‐CoA reductases from various organisms or the recently described acryloyl‐CoA reductase activity of propionyl‐CoA synthase from Chloroflexus aurantiacus.
Ionisation behaviour and solution properties of the potassium‐channel blocker ShK toxin
Authors: Tudor Jane E. , Pennington Michael W. , Norton Raymond S.
Published: March 2003

The effects of pH, temperature and polypeptide concentration on the solution structure and side chain interactions of ShK toxin, a potassium‐channel‐blocking polypeptide from the sea anemone Stichodactyla helianthus, have been investigated by means of one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. Resonance assignments have been obtained for most protons in the molecule, and for the α and β carbon atoms. The lack of concentration dependence of the 1H chemical shifts and linewidths indicates that self‐association is not significant and cannot account for the sheet‐like structure near the N terminus. The structure is stable to high temperature, showing little change even at 353 K. This stability allowed backbone‐amide temperature coefficients to be interpreted, and the correlation of these values with hydrogen bonds observed in the structures and with solvent exchange rates is discussed. pKa values have been measured for Asp5, His19 and Tyr23, and the contributions to these pKa values from other residues investigated using the analogues R11Q (denoting substitution of Arg11 with Gln), R11E, H19K, K22A, Y23A and K30A. These results show that Asp5 (pKa 2.8) makes an electrostatic interaction with Lys30, which may be partially responsible for the importance of these side chains in the folding of synthetic toxin. The phenolic pKa of Tyr23 is reduced to 8.7 in the native toxin, as a result of interactions with the positively charged side chains of Arg11 and to a lesser extent Lys22. Several hydrogen bonds between the Arg11 guanidino group and the Tyr23 phenolic group are found in the solution structures. As these three residues are implicated in the tight binding of ShK toxin to the T‐lymphocyte voltage‐gated potassium channel Kv1.3, their close interactions should be taken into account in models of binding of this toxin to the pore and vestibule of this and other potassium channels.
Structural and functional divergence of class II alcohol dehydrogenase
Authors: Svensson Stefan , Hedberg Jesper J. , Höög Jan‐Olov
Published: March 2003

cDNAs coding for class II alcohol dehydrogenase were isolated from a rabbit‐liver cDNA library. Deduced amino acid sequences show that isozymic forms of rabbit class II alcohol dehydrogenase exist, with a positional identity of 88.4 %. A high variability in structure of class II alcohol dehydrogenase between the species is also reflected in function. The rabbit II‐1 isozyme shows common characteristics with the human enzyme, but has a lower Km value for ethanol, 4.2 mM. The II‐2 isozyme shows restriction for aliphatic alcohols longer than pentanol. For shorter alcohols the II‐2 form has similar Km values as the II‐1 isozyme, 5.5 mM for ethanol, but is a low activity variant with a 10‐fold decrease in kcat values compared with II‐1. Nevertheless, II‐2 has a higher specificity for benzoquinone than II‐1 due to a lower Km value, 80 μM compared with 1 mM, and is in this sense more like the human class II enzyme. In addition a rabbit class III alcohol dehydrogenase cDNA was isolated that encodes a typical class III enzyme/glutathione‐dependent formaldehyde dehydrogenase.

The finding of isozymic forms of class II alcohol dehydrogenase is in line with the evolution of the system of medium‐chain alcohol dehydrogenases with different enzymes, different classes and different isozymes and further underline the complexity of the entire mammalian alcohol dehydrogenase system.
Two distinct actin‐binding sites of smooth muscle calponin
Authors: Mino Terumasa , Yuasa Uhito , Nakamura Fumiaki , Naka Michiko , Tanaka Toshio
Published: March 2003

Amino acid residues 145−163 of calponin have been proposed as a putative actin‐binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867−8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194−6201] enhanced the Ca2+‐induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20 400−20 403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172−His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172−His187 inhibited the binding of calponin to F‐actin in a concentration‐dependent manner but not the binding of caldesmon. Gly141−Gly160, including the above‐mentioned putative actin‐binding site, also competed with intact calponin to the same extent as Lys172−His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141−Gly160. Lys172−His187 and other tested peptides had no effect. However, Gly141−Gly160 and Lys172−His187 reduced the fluorescence intensity of pyrene‐labeled F‐actin with approximately equal potency. Moreover, Lys172−His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172−His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin‐binding activity. These observations suggest the direct involvement of two distinct actin‐binding sites, with different regulatory functions, in the interactions of calponin with actin.
1H‐NMR study of the structural influence of Y64 substitution in Desulfovibrio vulgaris Hildenborough cytochrome c553
Authors: Sebban‐Kreuzer Corinne , Blanchard Laurence , Bersch Beate , Blackledge Martin J. , Marion Dominique , Dolla Alain , Guerlesquin Françoise
Published: March 2003

Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical‐shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64←F substitution induces particular effects on the NH exchange at the N‐terminal, C‐terminal and central α‐helices and increases the stability of the oxidized molecule.
Delayed ontogenesis of CYP1A2 in the human liver
Authors: Sonnier Michelle , Cresteil Thierry
Published: March 2003

The ontogenesis of CYP1A proteins was investigated in a human liver bank composed of fetal, neonatal and adult samples. In immunoblots, a polyclonal antibody raised against rat CYP1A1, cross‐reacted with cDNA‐expressed human CYP1A1 and CYP1A2. In adult liver microsomes, this antibody reacted with a single band identified as the CYP1A2 protein, while no CYP1A1 could be detected. CYP1A2 protein was absent in microsomes prepared from fetal and neonatal livers and its levels increased in infants aged 1−3 months to attain 50 % of the adult value at one year. Enzymatic activities supported by CYP1A proteins were assayed on these samples. Methoxyresorufin demethylase supported by the CYP1A2 recombinant protein followed the same ontogenic profile as the CYP1A2 protein. In liver microsomes, the demethylation of imipramine was essentially due to CYP1A2 and to a smaller extent to CYP3A. In fetuses and early neonates, CYP3A proteins were responsible for the low demethylation of imipramine (3−4 % of the adult activity) before the onset of CYP1A2 and the subsequent rise of activity. Immunodetection and enzymatic activities were consistent with the absence of CYP1A1 and the late expression of CYP1A2 in the human liver, compared to the early rise of CYP3A4, CYP2C, CYP2D6, and CYP2E1 proteins.
Structural investigation of the exopolysaccharide produced by Pseudomonas flavescens strain B62
Authors: Cescutti Paola , Toffanin Renato , Fett William F. , Osman Stanley F. , Pollesello Piero , Paoletti Sergio
Published: March 2003

Pseudomonas flavescens strain B62 (NCPPB 3063) is a recently described bacterium isolated from walnut blight cankers. This strain has been designated as the type strain of a Pseudomonas rRNA groupI species. Strain B62 produced a mixture of two exopolysaccharides, differing in weight average relative molecular mass and composition. Only the most abundant exopolysaccharide (90 % by mass), corresponding to the one with the lower molecular mass, was investigated by use of methylation analysis, partial acid hydrolysis, and NMR spectroscopy. The polysaccharide was depolymerised by the action of the cellulase produced by Penicillum funiculosum and the oligosaccharide obtained, corresponding to the repeating unit, was characterised by NMR spectroscopy and ion‐spray mass spectrometry. The repeating unit of the B62 exopolysaccharide is
AcAc
∣∣
66
←4)‐β‐
D‐Xp‐(1←4)‐β‐
D‐Glcp‐(1←
3
↑
1
α‐
L‐Fucp
4
↑
1
4‐O‐
L‐Lac‐β‐
D‐GlcpA
where X is glucose (75 %) or mannose (25 %), and Lac is lactate. The O‐acetyl groups are present only on 75 % of the repeating units, and they are linked to the C6 of the hexose residues in non‐stoichiometric amounts.
The mechanism of porcine pancreatic α‐amylase
Authors: Al Kazaz Modhafar , Desseaux Véronique , Marchis‐Mouren Guy , Prodanov Eugenio , Santimone Marius
Published: March 2003

A kinetic study was carried out on the inhibitory effects of acarbose, maltose, and maltotriose on porcine pancreatic α‐amylase (PPA), using maltopentaose as the substrate.

Lineweaver‐Burk plots showed that the inhibitory action of acarbose is of the mixed non‐competitive type. The secondary plots gave straight lines. A model involving abortive complexes accounts for these results. Dixon plot analysis led to the same conclusion. According to the proposed model, one molecule of acarbose per amylase molecule binds either directly to free enzyme at the active site or to the enzyme‐substrate complex at a secondary carbohydrate‐binding site, which becomes functional after the substrate has bound to the enzyme molecule at the active site.

Kinetic analysis of the inhibition exerted by either the maltose or maltotriose reaction products of maltopentaose hydrolysis were then performed. The inhibitory effect of maltose was found to be of the non‐competitive type, while that of maltotriose was competitive. It can therefore be concluded that the first reaction product to be released upon maltopentaose hydrolysis is maltose, and that the second product is maltotriose. This indicates that after hydrolysis of the maltopentaose chain, the reducing side fragment is released first.
Identification of a specific effector of the small GTP‐binding protein Rap2
Authors: Janoueix‐Lerosey Isabelle , Pasheva Evdokia , de Tand Marie‐France , Tavitian Armand , de Gunzburg Jean
Published: March 2003

Rap2 is a small GTP‐binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two‐hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2‐interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled‐coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7 % identity in their common region. A search for similar sequences in expressed‐sequence‐tags databanks revealed the presence in human and rodents of mRNAs encoding the 400‐residue and 446‐residue forms of RPIP8. Furthermore a doublet of 45−50 kDa, corresponding to the 400‐residue and 446‐residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic β‐cell lines HIT‐T15 and RIN‐m5F. Using transient transfections of HIT‐T15 cells it was possible to demonstrate that [Val12]Rap2 and wild‐type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.
In vitro characterisation of the interaction between newly synthesised proteins and a pancreatic isoform of protein disulphide isomerase
Authors: Elliott John G. , Oliver Jason D. , Volkmer Jorg , Zimmermann Richard , High Stephen
Published: March 2003

The lumen of the endoplasmic reticulum (ER) contains an array of molecular chaperones and folding factors that modulate the folding and assembly of newly synthesised proteins entering the secretory pathway. One of these components, protein disulphide isomerase (PDI), facilitates the formation of the correct disulphide bonds within newly synthesised polypeptides, and is the archetype for a family of sequence related PDI‐like proteins. We have investigated the interaction between a recently identified, pancreas‐specific PDI‐like protein (PDIp), and in vitro synthesised secretory and membrane proteins produced in the presence of ER‐derived canine pancreatic microsomes. We have previously established that a second PDI‐like protein, ERp57, interacts specifically with N‐glycosylated proteins. In contrast, we find that the interaction of PDIp with newly synthesised proteins occurs independently of any requirement for N‐linked glycosylation. In this respect, the properties of PDIp mirror those of archetypal PDI. When the carbohydrate‐dependent interactions between glycoproteins and ERp57 are blocked by drug treatment, the association of these precursors with both PDIp and PDI is enhanced. We propose that PDI‐like proteins have overlapping specificity and may exhibit some degree of functional redundancy.
Atherogenic concentrations of native low‐density lipoproteins down‐regulate nitric‐oxide‐synthase mRNA and protein levels in endothelial cells
Authors: Vidal Francisco , Colomé Cristina , Martínez‐González José , Badimon Lina
Published: March 2003

The nitric oxide synthase family of proteins is the unique class of mammalian enzymes that metabolizes L‐arginine to form nitric oxide (NO). The atherogenic action of low‐density lipoproteins (LDL) may be mediated, in part, by its effects on endothelial‐derived nitric oxide. To determine whether native LDL (nLDL), at atherogenic concentrations, are capable of modulating NO synthase expression, we treated human umbilical vein endothelial cells with increasing concentrations of human nLDL (0−240 mg cholesterol/dl) for various time periods (2−48 h). Northern and western blot analyses indicate that both endothelial NO‐synthase mRNA and protein are down‐regulated by atherogenic concentrations of nLDL (180 and 240 mg cholesterol/dl) after 48 h of incubation. Cycloheximide and actinomycin D experiments suggest that this down‐regulation operates at a transcriptional level. Additionally, treatment of the cells with high‐density lipoproteins, at human physiological concentrations (45 mg cholesterol/dl), does not appear to alter the expression of endothelial NO synthase which seems to indicate that nLDL affect the gene transcription rate by a specific and concentration‐dependent mechanism. These findings may have important implications because they provide a novel mechanism by which hypercholesterolemia induces early changes on endothelial cells that could have pathophysiological significance in the atherosclerotic process.
Fatty acid elongation in yeast
Authors: Dittrich Franziska , Zajonc Dirk , Hühne Kathrin , Hoja Ursula , Ekici Arif , Greiner Eva , Klein Hans , Hofmann Jörg , Bessoule Jean‐Jacques , Sperling Petra , Schweizer Eckhart
Published: March 2003

Elongation of long‐chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl‐CoA, NADPH and a medium‐chain or long‐chain acyl‐CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12−14 carbon atoms, while shorter‐chain‐length acyl‐CoA were almost (octanoyl‐CoA) or completely (hexanoyl‐CoA, acetyl‐CoA) inactive. In particular, acetyl‐CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl‐CoA (0.33 mM), decanoyl‐CoA (0.83 mM) lauroyl‐CoA (0.05 mM), myristoyl‐CoA (0.4 mM) and palmitoyl‐CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl‐CoA was 17‐fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (  12 : 0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10−20‐fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15−17 or 22−26 carbon atoms, depending on whether tridecanoyl‐CoA or stearoyl‐CoA is used as a primer. In vitro, the elongation products are converted in part, by α‐oxidation, to their odd‐chain‐length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd‐chain‐length elongation products or very‐long‐chain fatty acids (VLCFA) shorter than 26 : 0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell homogenate. In addition, the in vivo process appears to be protected against side reactions such as hydrolysis or α‐oxidation. Yeast mutants defective in 12 : 0 or 13 : 0 elongation were derived from fas‐mutant strains according to their failure to grow on 13 : 0‐supplemented media. In vivo, 12 : 0 elongation was reduced to 0−10 % of the normal level, while 16 : 0 elongation and VLCFA synthesis were unimpaired. It is concluded that yeast contains either two different elongation systems, or that the respective mutation interferes differentially with medium‐chain and long‐chain fatty acid elongation. The yeast gene affected in the elongation‐defective mutants was isolated and, upon sequencing, identified as the known ELO1 sequence. It encodes a putative membrane protein of 32‐kDa molecular mass with no obvious similarity to any of the known FAS component enzymes.
Demonstration of a Ca2+ requirement for thyroglobulin dimerization and export to the golgi complex
Authors: Di Jeso Bruno , Pereira Rodolfo , Consiglio Eduardo , Formisano Silvestro , Satrustegui Jorgina , Sandoval Ignacio V.
Published: March 2003

We have examined the effects of depleting the endoplasmic reticulum Ca2+ store on the maturation of newly synthesized thyroglobulin molecules, their export to the Golgi complex, and their secretion by FRTL‐5 cells. An inhibitor of the endoplasmic reticulum Ca2+ pump, thapsigargin, and the Ca2+ ionophore A23187 depleted the endoplasmic reticulum Ca2+ store and strongly inhibited thyroglobulin secretion in cells chased in medium containing 0.1 mM Ca2+. Inhibition of thyroglobulin secretion was caused by a block in the export of newly synthesized thyroglobulin molecules from the endoplasmic reticulum to the Golgi complex, as shown by cell‐fractionation experiments and the intracellular accumulation of endoH‐sensitive thyroglobulin. The thyroglobulin molecules retained in the endoplasmic reticulum of cells treated with the drugs were found to assemble more slowly into dimers than thyroglobulin in control cells. Protease‐sensitivity experiments demonstrated that thyroglobulin dimers assembled in the presence of thapsigargin had a different conformation with respect to dimers assembled in controls cells.
Characterisation of a catabolic epoxide hydrolase from a Corynebacterium sp.
Authors: Misawa Elisa , Chan Kwo Chion Chan K. C. , Archer Ian V. , Woodland Marc P. , Zhou Ning‐Yi , Carter Semra F. , Widdowson David A. , Leak David J.
Published: March 2003

The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic‐interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32 140 Da. The gene encoding Corynebacterium EH was located on a 3.5‐kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N‐terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14 650−14 657), particularly around the catalytic site. All of these proteins belong to the α/β‐hydrolase‐fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.
Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23
Authors: Utkin Yuri N. , Krivoshein Arcadius V. , Davydov Vasily L. , Kasheverov Igor E. , Franke Peter , Maslennikov Innokenty V. , Arseniev Alexander S. , Hucho Ferdinand , Tsetlin Victor I.
Published: March 2003

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N‐hydroxysuccinimide esters of p‐azidobenzoic, p‐azidotetraflourobenzoic, p‐benzoylbenzoic and p‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzoic acids. The ion‐exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross‐linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross‐linking yield (up to 16 % of the bound toxin) was observed for azidobenzoyl‐Lys23‐CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group : the azido derivatives labelled the γ and δ subunits, benzoylbenzoyl derivative labelled the α and δ subunits, while p‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzoyl derivative reacted with α, γ and δ subunits.

The cross‐linking experiments in the presence of varying concentrations of (+)‐tubocurarine demonstrated that the Lys23‐attached diazirinyl group contacts the δ and α subunits in one ligand‐binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23‐diazirinyl are with γ and α subunits. This means that the central loop in the two CTX molecules binds at the α/γ and α/δ interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X‐ray structure of CTX, showed that this value does not exceed 13 Å. The results obtained favor the disposition of the ligand‐binding sites at the subunit interfaces, with the distance between α and δ, or α and γ subunits at these sites being not more than 13 Å.
Binding of the Co(NH3)4 derivative of (2′)3′‐O‐[N‐methyl‐anthraniloyl]‐ATP to the E2ATP site of Na+/K+‐transporting ATPase lowers the conformational flexibility of its E1ATP site
Authors: Schoner Wilhelm , Mertens Wolfgang , Helms Michael , Fortes George
Published: March 2003

Na+/K+‐activated ATP hydrolysis by the sodium pump is catalyzed by the interaction of high‐affinity and low‐affinity ATP‐binding sites [Thoenges, D. & Schoner, W. (1997) J. Biol. Chem. 272, 16 315−16 321]. To explore how binding of ATP to the low‐affinity E2ATP site affects the conformational flexibility of the high‐affinity E1ATP site due to interaction with Na+ or K+ ions, the E2ATP site was blocked with a fluorescent MgATP complex analog and fluorescence changes of the E1ATP site modified by FITC (fluorescein 5′‐isothiocyanate) were studied. The fluorescent MgATP complex analog Co(NH3)4MANT‐ATP [β,γ‐bidentate complex of 2′(3′)‐O‐(N‐methylanthraniloyl)‐ATP] inactivated Na+/K+‐ATPase in a time‐dependent and concentration‐dependent process with a Kd of 0.17 mM and an inactivation rate constant, k, of 0.031 min−1. ATP protected against the inactivation with a Kd of 0.43 mM. Consistent with a modification of the E2ATP binding site, Co(NH3)4MANT‐ATP inactivated the K+‐activated phosphatase activity in an enzyme whose E1ATP site had already been modified by FITC. Inactivation by Co(NH3)4MANT‐ATP was due to tight binding which resulted in a loss of fluorescence. Tightly bound Co(NH3)4MANT‐ATP could only be released by denaturation with SDS. Analysis of the conformational flexibility of the E1ATP site after labeling with FITC led to a K+‐dependent quench of fluorescence which is reversed by Na+. This flexibility was lost upon the blockade of the E2ATP site by Co(NH3)4MANT‐ATP.