bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific inhibition of barley α‐amylase 2 by barley α‐amylase/subtilisin inhibitor depends on charge interactions and can be conferred to isozyme 1 by mutation
Authors: Rodenburg Kees W. , Vallée Francois , Juge Nathalie , Aghajari Nushin , Guo Xiao‐jun , Haser Richard , Svensson Birte
Published: March 2003

α‐Amylase 2 (AMY2) and α‐amylase/subtilisin inhibitor (BASI) from barley bind with Ki = 0.22 nm. AMY2 is a (β/α)8‐barrel enzyme and the segment Leu116–Phe143 in domain B (Val89–Ile152), protruding at β‐strand 3 of the (β/α)8‐barrel, was shown using isozyme hybrids to be crucial for the specificity of the inhibitor for AMY2. In the AMY2–BASI crystal structure [F. Vallée, A. Kadziola, Y. Bourne, M. Juy, K. W. Rodenburg, B. Svensson & R. Haser (1998) Structure6, 649–659] Arg128AMY2 forms a hydrogen bond with Ser77BASI, while Asp142AMY2 makes a salt‐bridge with Lys140BASI. These two enzyme residues are substituted by glutamine and asparagine, respectively, to assess their contribution in binding of the inhibitor. These mutations were performed in the well‐expressed, inhibitor‐sensitive hybrid barley α‐amylase 1 (AMY1)‐(1–90)/AMY2‐(90–403) with Ki = 0.33 nm, because of poor production of AMY2 in yeast. In addition Arg128, only found in AMY2, was introduced into an AMY1 context by the mutation T129R/K130P in the inhibitor‐insensitive hybrid AMY1‐(1–161)/AMY2‐(161–403). The binding energy was reduced by 2.7–3.0 kcal·mol−1 as determined from Ki after the mutations R128Q and D142N. This corresponds to loss of a charged interaction between the protein molecules. In contrast, sensitivity to the inhibitor was gained (Ki = 7 µm) by the mutation T129R/K130P in the insensitive isozyme hybrid. Charge screening raised Ki 14–20‐fold for this latter mutant, AMY2, and the sensitive isozyme hybrid, but only twofold for the R128Q and D142N mutants. Thus electrostatic stabilization was effectively introduced and lost in the different mutant enzyme–inhibitor complexes and rational engineering using an inhibitor recognition motif to confer binding to the inhibitor mimicking the natural AMY2–BASI complex.
Functional characterization and mechanism of action of recombinant human kynurenine 3‐hydroxylase
Authors: Breton Jérôme , Avanzi Nilla , Magagnin Simona , Covini Nevie , Magistrelli Giovanni , Cozzi Liviana , Isacchi Antonella
Published: March 2003

The mitochondrial outer membrane enzyme kynurenine 3‐hydroxylase (K3H) is an NADPH‐dependent flavin mono‐oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS‐1 cells as a glutathione S‐transferase (GST) fusion protein, and the pure recombinant protein (rec‐K3H) was obtained with a specific activity of about 2000 nmol·min−1·mg−1. Rec‐K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non‐covalently bound FAD per molecule of enzyme. The mechanism of the rec‐K3H‐catalyzed reaction was investigated by overall initial‐rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl−, and that NADPH‐catalyzed oxidation occurred even in the absence of kynurenine if 3‐hydroxykynurenine was present, suggesting an uncoupling effect of 3‐hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3‐hydroxykynurenine in vivo.
Insertion of light‐harvesting chlorophyll a/b protein into the thylakoid
Authors: Kosemund Kirstin , Geiger Inke , Paulsen Harald
Published: March 2003

The major light‐harvesting chlorophyll a/b‐binding protein (Lhcb1,2) of photosystem II is inserted into the thylakoid via the signal recognition particle dependent pathway. However, the mechanism by which the protein enters the membrane is at this time unknown. In order to define some topographical restrictions for this process, we constructed several recombinant derivatives of Lhcb1 carrying hexahistidine tags at either protein terminus or in the stromal loop domain. Additionally, green fluorescent protein (GFP) was fused to either terminus. None of the modifications significantly impair the pigment‐binding properties of the protein in the in vitro reconstitution of LHCII. With the exception of the C‐terminal GFP fusion, all mutants stably insert into isolated thylakoids in the absence of Ni2+ ions. The addition of low concentrations of Ni2+ ions abolishes the thylakoid insertion of C‐terminally His‐tagged mutants whereas the other His‐tagged proteins fail to insert only at higher Ni2+ concentrations. The C‐terminus of Lhcb1 must cross the membrane during protein insertion whereas the other sites of Lhcb1 modification are positioned on the stromal side of LHCII. We conclude that a Ni2+‐complexed His tag and fusion to GFP inhibit translocation of the protein C‐terminus across the thylakoid. Our observations indicate that the N‐terminal and stromal domain of Lhcb1 need not traverse the thylakoid during protein insertion and are consistent with a loop mechanism in which only the C‐terminus and the lumenal loop of Lhcb1 are translocated across the thylakoid.
cDNA cloning, genomic cloning, and tissue‐specific regulation of mouse cerebroside sulfotransferase
Authors: Hirahara Yukie , Tsuda Masayuki , Wada Yoshinao , Honke Koichi
Published: March 2003

We have isolated a mouse cDNA clone encoding 3′‐phosphoadenylylsulfate–galactosylceramide 3′‐sulfotransferase (cerebroside sulfotransferase; CST; EC 2.8.2.11) from a kidney cDNA library, using a human CST cDNA clone [Honke, K., Tsuda, M., Hirahara, Y., Ishii, A., Makita, A. & Wada, Y. (1997) J. Biol. Chem.272, 4864–4868] as a probe. A recombinant protein of the cloned cDNA showed CST activity. The deduced protein is composed of the same 423 amino acids as human CST and its sequence exhibits 84% identity with that of the human counterpart. Northern‐blot analysis and subquantitative reverse transcription‐PCR (RT‐PCR) analysis showed that the CST gene is preferentially transcribed in stomach, small intestine, brain, kidney, lung, and testis, in that order. To examine differences in transcripts in various tissues, we isolated CST cDNA clones from stomach, small intestine, brain, kidney, and testis by 5′‐RACE analysis. We found seven different nucleotide sequences in the 5′‐UTR, while the DNA sequences of all the isolated cDNA clones were identical in the coding region. In addition, we isolated CST genomic DNA clones from a mouse genomic library. The clones covered all the 5′‐UTR sequences and coding exons including 3′‐UTR. RT‐PCR analyses of CST mRNAs from various tissues confirmed that CST transcripts are tissue‐specifically spliced by alternative use of multiple exons 1. These observations suggest that the tissue‐specific expression of the CST gene is explained by alternative usage of multiple 5′‐UTR exons flanked with tissue‐specific promoters.
Biochemical and functional characterization of the Tn‐specific lectin from Salvia sclarea seeds
Authors: Medeiros Andrea , Bianchi Sergio , Calvete Juan J. , Balter Henia , Bay Sylvie , Robles Ana , Cantacuzène Danièle , Nimtz Manfred , Alzari Pedro M. , Osinaga Eduardo
Published: March 2003

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcα‐O‐Ser/Thr), a specific marker of many human carcinomas. Two‐dimensional electrophoresis, amino‐acid and amino‐sugar analysis, and MALDI‐TOF MS showed that SSL is an acidic (pI 5.5), 60–61‐kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 ± 9 kDa. This value did not change in the pH range 2.5–8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N‐glycans released from SSL by hydrazinolysis indicated that SSL possesses 2–3 glycosylation sites occupied with the typical plant glycans Manα1–6[(Manα1–3)(Xylβ1–2)]Manβ1–4‐GlcNAcβ1–4(Fucα1–3)GlcNAc and [(Manα1–3/6)(Xylβ1–2)]Manβ1–4‐GlcNAcβ1–4(Fucα1–3)GlcNAc. The influence of adjacent Tn structures on the binding of two Tn‐specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti‐Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.
Structure–function relationships of temporins, small antimicrobialpeptides from amphibian skin
Authors: Mangoni M. Luisa , Rinaldi Andrea C. , Di Giulio Antonio , Mignogna Giuseppina , Bozzi Argante , Barra Donatella , Simmaco Maurizio
Published: March 2003

Temporins, antimicrobial peptides of 10–13 residues, were isolated from secretions of Rana temporaria[Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L. & Barra, D. (1996) Eur. J. Biochem.242, 788–792]. These molecules are specific to this amphibian species, which is also able to secrete on its skin other antimicrobial peptides similar to those found in different Rana species. The effect of temporins A, B and D (13 residues, net charge +2), and H (10 residues, net charge +1 and +2, respectively) against both artificial membranes of differing lipid composition and bacteria has been investigated in order to gain insight into their mechanisms of action. The results indicate that: the lytic activity of temporins is not greatly affected by the membrane composition; temporins A and B allow the leakage of large‐size molecules from the bacterial cells; temporin H renders both the outer and inner membrane of bacteria permeable to hydrophobic substances of low molecular mass; and temporin D, although devoid of antibacterial activity, has a cytotoxic effect on erythrocytes. The results allow important conclusions to be drawn about the minimal structural requirements for lytic efficiency and specificity of temporins.
Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins α‐ and γ‐SNAP (soluble N‐ethylmaleimide‐sensitive factor‐attachment proteins) families in Dictyostelium discoideum
Authors: Weidenhaupt Marianne , Bruckert Franz , Louwagie Mathilde , Garin Jérôme , Satre Michel
Published: March 2003

The soluble N‐ethylmaleimide‐sensitive‐factor‐attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in α/β and γ subfamilies. SNAPs act as adapters between N‐ethylmaleimide‐sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP‐driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c‐myc‐tagged form of D. discoideum NSF (NSF‐myc). Its immunoprecipitation from detergent‐solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF‐myc immunoprecipitates from cytosol. Analysis of trypsin‐digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of α‐ and γ‐SNAP, respectively. The α‐/γ‐SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of γ‐SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the γ subtype. Altogether, these results suggest a specific role for γ‐SNAP, distinct from that of α‐SNAP.
Purification of proctolin‐binding proteins from the foregut of the insect Blaberus craniifer
Authors: Mazzocco Claire , Puiroux Jacques
Published: March 2003

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin‐binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H‐proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five‐step chromatographic purification including chromatofocusing, anion‐exchange and size‐exclusion chromatographies. The final size‐exclusion separation resulted in the isolation of ≈ 100 pmol of purified proctolin‐binding proteins, eluting as a single peak at ≈ 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained ≈ 7–8 µg of protein, suggesting that one band corresponds to the proctolin‐binding activity. Proctolin‐binding proteins were thus purified 1800‐fold using standard chromatography.
Morphine‐like substance in leech ganglia
Authors: Laurent Virginie , Salzet Béatrice , Verger‐Bocquet Martine , Bernet François , Salzet Michel
Published: March 2003

Binding experiments followed by measurement of nitric oxide release revealed an opiate alkaloid high affinity receptor with no affinity to opioids, representing a new µ‐subtype receptor in the brain of the leech Theromyzon tessulatum. In addition, evidence of morphine‐like substances was found in immunocytochemical studies and HPLC coupled to electrochemical detection (500 mV and 0.02 Hz). Based on previous evidence of the involvement of morphine as an immune response inhibitor, we demonstrate that in leech ganglia injection of lipopolysaccharide (LPS; a potent immunostimulatory agent derived from bacteria) provoked an increase in the level of ganglionic morphine‐like substances after a prolonged latency period of 24 h (from 2.4 ± 1.1 pmol per ganglion to 78 ± 12.3 pmol per ganglion; P < 0.005; LPS injected 1 µg·mL−1); this effect is both concentration‐ and time‐dependent. Finally, we have demonstrated that morphine, after binding to its own receptor, inhibits leech immunocyte activation through adenylate cyclase inhibition and nitric oxide release. This report confirms that morphine is an evolutionarily stable potent immunomodulator.
Digestion of single crystals of mannan I by an endo‐mannanase from Trichoderma reesei
Authors: Sabini Elisabetta , Wilson Keith S. , Siika‐aho Matti , Boisset Claire , Chanzy Henri
Published: March 2003

The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a β‐mannanase (EC 3.2.1.78 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo‐mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.
Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody
Authors: Demangel Caroline , Maroun Rachid C. , Rouyre Sylvie , Bon Cassian , Mazié Jean‐Claude , Choumet Valérie
Published: March 2003

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three‐chain subunit, CA, which increases the lethal potency of CB. The A‐56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A‐56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A‐56.36 and CB had overlapping binding sites on CA. By screening random phage‐displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A‐56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin‐dissociating effect of A‐56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A‐56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage‐displayed peptides corresponding to fragments of the two identified regions on CA reacted with A‐56.36 and with CB. Our data support the hypothesis that A‐56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA–CB complex formation.
Domain organization of p130, PLC‐related catalytically inactive protein, and structural basis for the lack of enzyme activity
Authors: Kanematsu Takashi , Yoshimura Kenji , Hidaka Kiyoshi , Takeuchi Hiroshi , Katan Matilda , Hirata Masato
Published: March 2003

The 130‐kDa protein (p130) was isolated as a novel inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]‐binding protein similar to phospholipase C‐δ1 (PLC‐δ1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem.267, 6518–6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J.313, 319–325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC‐δ1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC‐δ1 and demonstrating that all domains of p130, including the unique region at the C‐terminus, are stable, tightly folded structures. p130 truncated at either or both the N‐terminus (94 amino acids) and C‐terminus (851–1096 amino acids) expressed in COS‐1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC‐δ1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC‐δ1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.
Identification of ubiquitinated repeats in human erythroid α‐spectrin
Authors: Galluzzi Luca , Nicolas Gaël , Paiardini Mirko , Magnani Mauro , Lecomte Marie‐Christine
Published: March 2003

The spectrin role(s) is (are) very important for the shape and the physical properties of red cells, such as deformability and resistance to mechanical stresses. Moreover a variety of spectrin diseases are known. We have previously demonstrated [Corsi, D., Galluzzi, L., Crinelli, R. & Magnani, M. (1995) J. Biol. Chem.270, 8928–8935] that human erythroid α‐spectrin is ubiquitinated in vitro and in vivo. In order to define the ubiquitinated repeats of this long protein and find out a possible function, we have produced recombinant peptides encompassing the αIII‐, αIV‐, αV‐ and EF hand domains of α‐spectrin chain. These peptides were tested in in vitro ubiquitin conjugation assays and two regions susceptibles to ubiquitination were found. The first one, in the αIV‐domain, includes the repeat 17 and the second one, in the αV‐domain, includes the repeat 20 and a part of repeat 21. We also demonstrated that the susceptibility to ubiquitination of the αV‐domain is reduced by interaction with the corresponding portion of β‐spectrin chain (βIV‐domain). Thus, at least ubiquitination of αV‐domain is susceptible to cytoskeleton assembly and spectrin dimerization.
Biochemical characterization of MI‐ENG1, a family 5 endoglucanase secreted by the root‐knot nematodeMeloidogyne incognita
Authors: Béra‐Maillet Christel , Arthaud Laury , Abad Pierre , Rosso Marie‐Noëlle
Published: March 2003

A β‐1,4‐endoglucanase named MI‐ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root‐knot nematodeMeloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI‐ENG1 shares many enzymatic properties with known endoglucanases from plants, free‐living or rumen‐associated microorganisms and phytopathogens. In spite of the presence of a cellulose‐binding domain at the C‐terminus, the ability of MI‐ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant–nematode interaction.
Detection of modifications in the glucose metabolism induced by genetic mutations in Saccharomyces cerevisiae by 13C‐ and 1H‐NMR spectroscopy
Authors: Herve Martine , Buffin‐Meyer Bénédicte , Bouet Françoise , Tran‐Dinh Son
Published: March 2003

NMR spectroscopy may offer a suitable technique to characterize the glucose metabolism in response to genetic mutations in cells. The effects of various genetic modifications in Saccharomyces cerevisiae yeast were investigated using 13C‐ and 1H‐NMR spectroscopy associated with biochemical techniques. Cells were incubated with [1‐13C]glucose in order to study glucose consumption and the formation of various end‐products (ethanol, trehalose, glycerol, glutamate and amino acids) as a function of time.

Two types of genetic modifications were studied in S. cerevisiae. A genetic modification deleted the N‐terminal part of the TFC7 protein which is the smallest subunit (tau55) of the TFIIIC transcription factor. One secondary effect of this mutation was a large deletion of mitochondrial DNA giving the rho‐phenotype. The other genetic modification corresponded to the disruption of the HUF gene; the mutated cells were rho+ like the reference strain. Both mutations increase the glycolysis rate and glycerol synthesis and decrease trehalose production. The most modified cells, which contain both TFC7 deletion and HUF gene disruption, utilize glucose in the most extreme manner as in these cells the largest production of the two glycolytic products (ethanol and glycerol) and the smallest trehalose formation occur. The HUF gene disruption serves as a positive modulator of glycolysis and respiration. However, the TFC7 deletion, associated with the phenotype rho‐, induces the most damage in the cellular function, dramatically altering the behaviour of the Krebs cycle. The cycle becomes blocked at the level of 2‐oxoglutarate, detected by a characteristic pattern of the 13C‐NMR glutamate spectra. These NMR spectra corroborate the phenotypic data, the rho‐phenotype corresponding to deletions of mitochondria DNA which block all mitochondria protein synthesis and render the cells unable to derive energy from respiration. Moreover, as a consequence of the Krebs cycle blocking, alanine formation is also observed.
Energetics of myo‐inositol hexasulfate binding to human acidic fibroblast growth factor
Authors: Guzmán‐Casado Mercedes , Sánchez‐Ruiz José M. , El Harrous Mohamed , Giménez‐Gallego Guillermo , Parody‐Morreale Antonio
Published: March 2003

The binding of myo‐inositol hexasulfate to an N‐terminal truncated 132‐amino‐acid human acidic fibroblast growth factor form was studied by isothermal titration calorimetry. The technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change can also be calculated. Experiments in different buffers and pH values show that the proton balance in the reaction is negligible. Experiments at pH 7.0 in the presence of 0.2–0.6 m NaCl showed that the enthalpy and Gibbs energy changes parallel behaviour with ionic strength change, with values in the −21 to −11 kJ·mol−1 range in the first case and in the −31 to −22 kJ·mol−1 range in the second. No dependence of entropy on ionic strength was found, with a constant value of ≈ 35 J·K−1·mol−1 at all ionic strengths studied. The results can be interpreted in molecular terms by a model in which competitive binding of 3–4 chloride ions to the myo‐inositol‐binding site is assumed. Isothermal titration calorimetry was also performed at different temperatures and yielded a value of −142 ± 13 J·K−1·mol−1 for the heat‐capacity change at pH 7.0 and 0.4 m NaCl. Using different parametric equations in the literature, changes on ligand binding in the range −100 to −200 Å2 in solvent‐accessible surface areas, both polar and apolar, were calculated from thermodynamic data. These values suggest a negligible overall conformational change in the protein when the ligand binds and agree closely with calculations performed with NMR structural data, in which it is shown that the most important negative change in total solvent‐accessible surface area occurs in the amino acids Ile56, Gln57, Leu58 and Leu149, in the high‐affinity receptor‐binding region of the protein.
Keratan sulfates from bovine tracheal cartilage
Authors: Huckerby Thomas N. , Lauder Robert M.
Published: March 2003

Intact keratan sulfate chains derived from bovine tracheal cartilage have been examined using both one‐dimensional methods and the two‐dimensional experiments COSY‐45 and TOCSY for homonuclear shift correlations and a modified COLOC (correlated spectroscopy for long‐range couplings) approach for 13C–1H shift correlations. Partial 1H and 13C NMR signal assignments for residues within the intact polymer chain are reported; data derived from the repeat region signals and from chain cap residues are assigned by comparison with published data derived from oligosaccharides obtained through cleavage of keratan sulfate polymer chains using keratanase and keratanase II and are discussed in detail. The one‐dimensional spectra for both 1H and 13C nuclei contain highly crowded signal clusters for which data analysis is not directly possible. COSY‐45 analysis allow the correlation and assignment of many proton resonances located within the 3.4–4.8 p.p.m. chemical shift region while from the C/H correlation spectrum data are assignable for some signals within the complex set of carbon resonances which fall in the region between 68 and 86 p.p.m., This work using material from tracheal cartilage has permitted the first detailed combined 1H and 13C NMR examination of the primary keratan sulfate polymer structure; this sequence forms the basis for the more complex members of the keratan sulfate family present in other tissues such as articular cartilage and cornea where further residues such as (α1–3)‐linked fucose and (α2–6)‐linked N‐acetylneuraminic acid are also present. This nondestructive method of analysis complements the currently available degradative methods for structure determination which may then subsequently be utilized.
Structural requirements for thymosin β4 in its contact with actin
Authors: Simenel Catherine , Van Troys Marleen , Vandekerckhove Joël , Ampe Christophe , Delepierre Muriel
Published: March 2003

We examined the conformational preferences of mutants of thymosin β4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild‐type thymosin β4 conformation consists of an α‐helix (helix I) extending from residues 5–16 with a more stable fragment from lysine 11 to lysine 16 and a second α‐helix (helix II) encompassing residues 31–39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17–22) that form the two main entities of interaction with the actin molecule. The α‐1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C‐terminal end over the LKK‐segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin β4 binding entities required for full activity.
Zwitterionic and acidic glycosphingolipids of the Drosophila melanogaster embryo
Authors: Seppo Antti , Moreland Margaret , Schweingruber Hans , Tiemeyer Michael
Published: March 2003

Defining glycosphingolipid structures in species amenable to genetic manipulation, such as Drosophila melanogaster, provides a foundation for investigating mechanisms that regulate glycolipid expression. Therefore, eight of the 12 major glycosphingolipids, accounting for 64% of lipid‐linked carbohydrate in Drosophila embryos, were purified after separation into acidic and zwitterionic pools. The zwitterionic lipids possess phosphoethanolamine (PEtn) linked to one or more GlcNAc residues and comprise a family of serially related structures. The longest characterized glycolipid, an octaosylceramide, designated Nz28, has the structure: GalNAcβ,4(PEtn‐6)GlcNAcβ,3Galβ,3GalNAcα,4GalNAcβ,4(PEtn‐6)GlcNAcβ,3Manβ,4GlcβCer. Heptaosyl (Nz7), hexaosyl (Nz6), pentaosyl (Nz5) and tetraosyl (Nz4) forms of Nz28, sequentially truncated from the nonreducing terminus, possess only one PEtn moiety. The major acidic lipid, designated Az29, possesses two PEtn moieties and a glucuronic acid linked to a Gal‐extended Nz28. Two other acidic glycolipids, Az9 and Az6, exhibit one PEtn moiety and the same hexose and N‐acetylhexosamine composition as Az29 and Nz6, respectively. The fully extended Drosophila core oligosaccharide differs from that of other dipterans in the linkage at a single glycosidic bond, a distinction with significant structural and biosynthetic consequences. Furthermore, acidic species account for a larger proportion of total glycosphingolipid, and PEtn substitution of GlcNAc is more complete in the Drosophila embryo. Divergent characteristics may reflect interspecies variation or stage‐specific glycosphingolipid expression in dipterans.
Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols
Authors: Jorasch Petra , Warnecke Dirk C. , Lindner Buko , Zähringer Ulrich , Heinz Ernst
Published: March 2003

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis[Jorasch, P., Wolter, F.P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol.29, 419–430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities.

We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin‐layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyrano‐syl]‐sn‐glycerol, 1,2‐diacyl‐3‐[O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl‐(1→6)‐O‐β‐d‐glucopyranosyl]‐sn‐glycerol, sn‐3′‐[O‐β‐d‐glucopyranosyl]‐phosphatidylglycerol and sn‐3′‐[O‐(6′″‐O‐acyl)‐β‐d‐glucopyranosyl‐(1′′′→6′′)‐O‐β‐d‐glucopyranosyl]‐sn‐2′‐acyl‐phospha‐tidylglycerol. A 1,2‐diacyl‐3‐[O‐β‐d‐galactopyranosyl]‐sn‐glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana.

The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C‐labeled sugar acceptors and unlabeled UDP‐glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP‐glucose:1,2‐diacylglycerol‐3‐β‐d‐glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.
cDNA cloning of bradykinin‐potentiating peptides–C‐type natriuretic peptide precursor, and characterization of the novel peptide Leu3‐blomhotin from the venom of Agkistrodon blomhoffi
Authors: Murayama Nobuhiro , Michel Gilles H. , Yanoshita Ryohei , Samejima Yuji , Saguchi Kenichi , Ohi Hiroaki , Fujita Yoshiaki , Higuchi Shigesada
Published: March 2003

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino‐acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3‐blomhotin are located in the N‐terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin‐potentiating peptide. In the C‐terminal region, the sequence for the C‐type natriuretic peptide was located along with a preceding processing signal. The deduced amino‐acid sequences for the four bradykinin‐potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3‐blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3‐blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3‐blomhotin retained activity to inhibit the angiotensin‐converting enzyme.
Regulation of the expression of human ferrochelatase by intracellular iron levels
Authors: Taketani Shigeru , Adachi Yasushi , Nakahashi Yoshitsugu
Published: March 2003

Mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of a ferrous ion into protoporphyrin and contains a labile [2Fe−2S] cluster center at the C‐terminus. To clarify the roles of the iron–sulfur cluster in the expression of mammalian ferrochelatase, enzyme activity in human erythroleukemia K562 cells under iron‐depleted conditions was examined. Treatment of cells with an iron chelator, desferrioxamine, resulted in a decrease in enzyme activity, in a dose‐ and time‐dependent manner. Heme content decreased during desferrioxamine treatment of the cells. Addition of ferric ion‐nitrilotriacetate [Fe (III)NTA] to desferrioxamine‐containing cultures led to restoration of the reduction in the enzyme activity. While RNA blots showed that the amount of ferrochelatase mRNA remained unchanged during these treatments, the amount of ferrochelatase decreased with a concomitant decrease in enzyme activity. When full‐length human ferrochelatase was expressed in Cos7 cells, the activity was found mainly in the mitochondria and was decreased markedly by treatment with desferrioxamine. The activity in Cos7 cells expressing human ferrochelatase in cytoplasm decreased with desferrioxamine, but to a lesser extent. When Escherichia coli ferrochelatase, which lacks the iron–sulfur cluster, was expressed in Cos7 cells, the activity did not change following any treatment. Conversely, the addition of Fe (III)NTA to the culture of K562 and Cos7 cells led to an increase in ferrochelatase activity. These results indicate that the expression of mammalian ferrochelatase is regulated by intracellular iron levels, via the iron–sulfur cluster center at the C‐terminus, and this contributes to the regulation of the biosynthesis of heme at the terminal step.
Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition
Authors: Mounier Carine M. , Luchetta Philippe , Lecut Christelle , Koduri Rao S , Faure Grazyna , Lambeau Gérard , Valentin Emmanuel , Singer Alan , Ghomashchi Farideh , Béguin Suzette , Gelb Michael H. , Bon Cassian
Published: March 2003

Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q mutant prolong the lag time of thrombin generation in human platelet‐rich plasma with similar efficiency, indicating that hGIIA exerts an anticoagulant effect independently of phospholipid hydrolysis under ex vivo conditions. Charge reversal of basic residues on the interfacial binding surface (IBS) of hGIIA leads to decreased ability to inhibit prothrombinase activity, which correlates with a reduced affinity for factor Xa, as determined by surface plasmon resonance. Mutation of other surface‐exposed basic residues, hydrophobic residues on the IBS, and His48, does not affect the ability of hGIIA to inhibit prothrombinase activity and bind to factor Xa. Other basic, but not neutral or acidic, mammalian secreted phospholipases A2 (sPLA2s) exert a phospholipid‐independent inhibitory effect on prothrombinase activity, suggesting that these basic sPLA2s also bind to factor Xa. In conclusion, this study demonstrates that the anticoagulant effect of hGIIA is independent of phospholipid hydrolysis and is based on its interaction with factor Xa, leading to prothrombinase inhibition, even under ex vivo conditions. This study also shows that such an interaction involves basic residues located on the IBS of hGIIA, and suggests that other basic mammalian sPLA2s may also inhibit blood coagulation by a similar mechanism to that described for hGIIA.
Organization‐dependent effects of toxic bivalent ions
Authors: Liliom Károly , Wágner Gábor , Pácz Anita , Cascante Marta , Kovács János , Ovádi Judit
Published: March 2003

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO2–4 inhibit the tubulin polymerization at an IC50 of 14–24 µm with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 µm. BIAcore measurements for tubulin–tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross‐linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO42–. No inhibitory effect of Cu2+ or CrO2–4 on microtubule assembly was detected in the MAP‐containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO2–4 ions contain aggregates of thread‐like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10–14 µm with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.
Purification and partial characterization of the hydroxylase component of the methanesulfonic acid mono‐oxygenase from Methylosulfonomonas methylovora strain M2
Authors: Reichenbecher Wolfram , Murrell J. Colin
Published: March 2003

The reductase enzyme and the hydroxylase enzyme of the three‐component methanesulfonic acid mono‐oxygenase (MSAMO) from Methylosulfonomonas methylovora were purified. Purification of the reductase from M. methylovora using a range of chromatographic techniques was accompanied by complete loss of activity. Expression of the reductase as a glutathionine S‐transferase fusion protein in Escherichia coli cells was successful as judged from the size of the polypeptide band obtained on induction with isopropyl thio‐β‐d‐galactoside. Subsequent affinity purification of the fusion protein, however, led to a protein extract containing only glutathionine S‐transferase protein, indicating that the fusion protein was unstable in vitro. The hydroxylase component of the MSAMO was purified from M. methylovora to near electrophoretic homogeneity using Q‐Sepharose, hydroxyapatite and Mono Q chromatography. SDS/PAGE of the purified hydroxylase showed a single band at ≈ 43.7 kDa for the α‐subunit and a double band at ≈ 23 kDa for the β‐subunit. MS scans obtained with matrix‐assisted laser desorption/ionization and electrospray ionization showed single peaks for both subunits, with a mass of 48 145.4 Da for α, 20 479.1 Da for β, and 68 624.5 for the αβ‐monomer. Gel filtration revealed a mass of 209 kDa, suggesting an α3β3 structure for the native enzyme. Purified hydroxylase enzyme exhibited absorbance maxima at 330 nm, 460 nm and 570 nm, indicating the presence of iron‐sulfur centres. The protein preparations contained 1 mol sulfide and 3–4 mol iron per mol αβ‐monomer. Chromium, cobalt, copper, lead, nickel, molybdenum, tungsten and vanadium were not found. Flavins were also absent. Antibodies raised against the native hydroxylase enzyme cross‐reacted with cell‐free extract from M. methylovora cells grown with methanesulfonate, but not with extract from cells grown with methanol, confirming that MSAMO was specifically induced during growth on methanesulfonate.
The structure–function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense
Authors: Vedrenne Cécile , Bringaud Frédéric , Barrett Michael P. , Tetaud Emmanuel , Baltz Théo
Published: March 2003

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino‐acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Kmd‐glucose = 41 µm versus 64 µm). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Kid‐glucose = 0.8 mm). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino‐acid residues important for d‐glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C‐terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.
Intracellular inhibition of blood group A glycosyltransferase
Authors: Laferté Suzanne , Chan Nora W. C. , Sujino Keiko , Lowary Todd L , Palcic Monica M
Published: March 2003

We report the intracellular inhibition of blood group AN‐acetylgalactosaminyltransferase in the human colorectal carcinoma cell line HT29 by 3‐amino‐3‐deoxy‐[Fucα(1–2)]Galβ‐O(CH2)7CH3. Inhibition was demonstrated with a novel capillary electrophoresis assay that monitored decreased intracellular conversion of fluorescently labelled Fucα(1–2)Gal‐R acceptor to the corresponding A epitope, GalNAcα(1–3)[Fucα(1–2)]Galβ‐R. Growth of HT29 cells with either the amino‐inhibitor or a competitive substrate, Fucα(1–2)Galβ‐O(CH2)7CH3, also resulted in decreased expression of blood group A determinants on cell‐associated glycoproteins, as detected by immunoprecipitation analysis using A‐specific monoclonal antibodies. Furthermore, exposure of these cells to the amino‐inhibitor or competitive substrate resulted in significant reduction of cell‐surface expression of blood group A determinants. As integrin α3β1, a cell‐surface receptor mediating cell–cell and cell–extracellular matrix interactions, was shown previously to be a major carrier of blood group A determinants on HT29 cells, the studies described herein highlight the potential usefulness of these compounds for elucidating the role of blood group A determinants in biological phenomena.
Echistatin inhibits pp125FAK autophosphorylation, paxillin phosphorylation and pp125FAK–paxillin interaction in fibronectin‐adherent melanoma cells
Authors: Della Morte Rossella , Squillacioti Caterina , Garbi Corrado , Derkinderen Pascal , Belisario Maria A , Girault Jean‐Antoine , Di Natale Paola , Nitsch Lucio , Staiano Norma
Published: March 2003

Echistatin, a snake‐venom RGD‐containing protein, was previously shown to disrupt cell‐matrix adhesion by a mechanism that involves the reduction of pp125FAK tyrosine phosphorylation levels. The aim of this study was to establish the sequence of events downstream pp125FAK dephosphorylation that could be responsible for echistatin‐induced disassembly of actin cytoskeleton and focal adhesions in fibronectin‐adherent B16‐BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosphorylation and kinase activity of pp125FAK. One hour of cell exposure to echistatin caused a 39% decrease of pp125FAK Tyr397 phosphorylation and a 31% reduction of pp125FAK autophosphorylation activity as measured by immune‐complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125FAK. Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both paxillin and pp125FAK from focal adhesions. The reduction of paxillin phosphorylation may represent a critical step in the pathway by which disintegrins exert their biological activity, including the inhibition of experimental metastasis in vivo.