bluuebunny/crossref_metadata_2025 huggingface dataset:
Residues involved in co‐factor and substrate binding of the short‐chain dehydrogenase/reductase PTR1 producing methotrexate resistance in Leishmania
Authors: Leblanc Éric , Papadopoulou Barbara , Bernatchez Chantale , Ouellette Marc
Published: March 2003

The Leishmania short‐chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate‐resistant Leishmania mutants. PTR1 is active as a tetramer and can reduce pterins and folates. PTR1 has several of the hallmarks of the short chain dehydrogenase/reductase family, including a glycine rich co‐factor binding site at its N‐terminus, and the consensus catalytic site TyrXaa3Lys. To start probing the structure/function of PTR1, we have generated by site‐directed mutagenesis five mutants, either in the co‐factor−binding site, Y38D or in the catalytic site Y195F, Y195W, K199R or in a PTR1‐specific region, Y175F. The mutated versions of PTR1 were studied in vivo in Leishmania and at the biochemical level using purified proteins. The Y175F mutant showed properties similar to wild‐type PTR1 in every aspect tested, but all the other mutants were inactive even if they were purified as tetramers. To test the ability of the mutated PTR1 versions to bind its co‐factor and substrates, trypsin digestion experiments were carried out under conditions upon which binding will prevent wild‐type PTR1 of being digested by trypsin. The wild‐type PTR1 as well as Y175F and Y195F mutants were protected against trypsin digestion whereas Y38D and K199R mutants were not. Mutations in regions involved in co‐factor binding (Y38D) or in catalytic site (K199R) altered the binding of the ligands, explaining why those protein versions are inactive.
Bound water in apo and holo bovine heart fatty‐acid‐binding protein determined by heteronuclear NMR spectroscopy
Authors: Mesgarzadeh Azita , Pfeiffer Stefania , Engelke Jan , Lassen Dirck , Rüterjans Heinz
Published: March 2003

Two‐ and three‐dimensional heteronuclear NMR experiments have been performed to identify internally bound water molecules in the solution structure of bovine heart fatty‐acid‐binding protein (heart FABP). NOE and rotating‐frame NOE (ROE) cross peaks between protein protons and protons of bound water molecules were observed in two‐dimensional H2O‐ROE/NOE‐1H,15N‐heteronuclear single quantum coherence spectra recorded from a uniformly 13C/15N‐enriched sample of bovine heart FABP. Contacts between water protons and 23 NH protons of the protein backbone were identified. The protein structure consists of 10 antiparallel β‐strands (βA−βJ), forming two nearly orthogonal β‐sheets, and a short helix‐turn‐helix motif connecting β‐strands A and B. The spatial folding resembles a β‐barrel. Most of the water molecules are localized in the gap between β‐strands D and E, and near the two α‐helices. In the delipidated heart FABP additional contacts between water molecules and NH protons could be observed using a three‐dimensional rotating frame Overhauser 1H,15N heteronuclear single quantum coherence experiment obtained with a 15N‐labeled sample of apo‐heart FABP.
Studies on the effect of fucosylated and non‐fucosylated finger/growth‐factor constructs on the clearance of tissue‐type plasminogen activator mediated by the low‐density‐lipoprotein‐receptor‐related protein
Authors: Camani Chantal , Gavin Olivier , Bertossa Carole , Samatani Emmanuel , Kruithof Egbert K. O.
Published: March 2003

Previously we observed that the finger/growth factor (FG) region of tissue‐type plasminogen activator (t‐PA) blocked the low‐density‐lipoprotein‐receptor‐related protein (LRP)‐mediated clearance of t‐PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t‐PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t‐PA by human hepatoma cells. At position 83 it had a serine, whereas human t‐PA has a free cysteine and rodent t‐PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2‐Fc). Two variants were studied, one having Ser83, the other Arg83.

The two fucosylated FG2‐Fc chimeras were compared with each other, with non‐fucosylated FG and with intact t‐PA with regard to their effect on the clearance of t‐PA and t‐PA plasminogen‐activator inhibitor type 1 (PAI‐1). For this comparison, LRP‐specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF‐1) the clearance of t‐PA and of t‐PA PAI‐1 was inhibited more than 95 % by receptor‐associated protein, an inhibitor of LRP‐mediated clearance, whereas no t‐PA or t‐PA PAI‐1 clearance was observed in LRP‐deficient PEA‐13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2‐Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t‐PA and t‐PA PAI‐1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non‐fucosylated FG (IC50 1950 nM and 1560 nM) and 75‐fold lower than that of intact t‐PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP‐mediated clearance of t‐PA.
Phenylglyoxylate : NAD+ oxidoreductase (CoA benzoylating), a new enzyme of anaerobic phenylalanine metabolism in the denitrifying bacterium Azoarcus evansii
Authors: Hirsch Wolfram , Schägger Hermann , Fuchs Georg
Published: March 2003

Phenylglyoxylate (benzoylformate) is an intermediate in the anoxic metabolism of phenylalanine and phenylacetate. It is formed by α‐oxidation of phenylacetyl‐CoA. Phenylglyoxylate is oxidatively decarboxylated by phenylglyoxylate‐oxidoreductase to benzoyl‐CoA, a central intermediate of anaerobic aromatic metabolism. The phenylglyoxylate oxidizing enzyme activity in the denitrifying bacterium Azoarcus evansii was induced during anaerobic growth with phenylalanine, phenylacetate and phenylglyoxylate, but not with benzoate. The new enzyme phenylglyoxylate : acceptor oxidoreductase was purified and studied. The oxygen‐sensitive enzyme reduced both NAD+ and viologen dyes. It was composed of five subunits of approximately 50, 48, 43, 24, and 11.5 kDa; the native mass as determined by gel filtration was 370 kDa, suggesting an α2β2γ2δ2ε2 composition. Phenylglyoxylate : acceptor oxidoreductase exhibited an ultraviolet/visible spectrum characteristic for an iron‐sulfur protein and contained 35 ± 4 mol Fe, 36 ± 4 mol acid‐labile sulfur, and 1.1 ± 0.2 mol FAD/mol. The enzyme was specific for phenylglyoxylate (Km 45 μM) and coenzyme A (Km 55 μM); 2‐oxoisovalerate was oxidized with 15 % of the rate. The turnover number with benzyl viologen at 37 +C was 46 s−1 at the optimal pH of 8. The enzyme catalyzed a NAD(P)H :viologen dye transhydrogenation reaction, NAD(H) being the preferred coenzyme. It also catalyzed an isotope exchange between CO2 and the carboxyl group of the substrate. The data are consistent with the following hypothesis. The enzyme complex consists of a core enzyme of four subunits with the composition α2β2γ2δ2, as reported for archaeal 2‐oxoacid : ferredoxin oxidoreductases; this complex is able to reduce viologen dyes. The holoenzyme contains in addition an ε2 unit that catalyzes the transfer of electrons from a small ferredoxin‐like subunit of the core complex to NAD+; this unit also catalyzes the transhydrogenase reaction, carries FAD and resembles ferredoxin : NAD(P)+‐oxidoreductase.
A multistep process is responsible for product‐induced inactivation of glucose‐fructose oxidoreductase from Zymomonas mobilis
Authors: Fürlinger Monika , Haltrich Dietmar , Kulbe Klaus D. , Nidetzky Bernd
Published: March 2003

Glucose‐fructose oxidoreductase from the bacterium Zymomonas mobilis catalyzes a transhydrogenation reaction in which
D‐fructose reduction to
D‐sorbitol is coupled to the oxidation of
D‐glucose or other aldoses to the corresponding aldonolactones. Tightly protein‐bound NADP(H) serves as the cofactor. We found that the interaction of glucose‐fructose oxidoreductase with its aldonolactone product triggered a sequential process that affects the protein structure conformationally and chemically and, ultimately, results in an irreversible loss of enzyme activity. (1) Probably as a mechanistic requirement during the catalytic cycle, conformational realignments in glucose‐fructose oxidoreductase are induced by binding of the lactone and are manifested by a 1.7‐fold increased accessibility to iodide quenching of the fluorescence of the active‐site‐bound NADPH, the exposure of one reactive cysteine (likely Cys127) and strongly red‐shifted tryptophan fluorescence. (2) As a fast subsequent reaction in vitro, the cysteine residue is deactivated, thus leading to a local, structural destabilization of glucose‐fructose oxidoreductase that, without affecting enzyme activity, leads to twofold tryptophan fluorescence as well as the exposure of three further cysteine residues. (3) The completed deactivation of these cysteines is accompanied by a twofold increase in hydrophobic surface and thus aggregation of the glucose‐fructose oxidoreductase tetramer. Aggregation, but not release of the tightly bound NADP(H), ultimately leads to the loss of activity and completes the inactivation of glucose‐fructose oxidoreductase. Apparently small conformational changes at the NADP(H)‐binding site of glucose‐fructose oxidoreductase trigger high‐order protein associations and seem to be thus responsible for an incorrect oligomerization of the enzyme.
A detailed analysis of the C5a anaphylatoxin effector domain : selection of C5a phage libraries on differentiated U937 cells
Authors: Hennecke Meike , Otto Annette , Baensch Melanie , Kola Axel , Bautsch Wilfried , Klos Andreas , Köhl Jörg
Published: March 2003

We have used a phage‐display‐based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69−73 of hC5a without C‐terminal Arg74 (des‐Arg74‐C5a) were randomly mutated. In more than 80 % (position 72) or 90 % (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90 % of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70−72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4−10‐fold higher binding affinity as compared to des‐Arg74‐[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage‐displayed C5a was proven to be a powerful strategy to define receptor‐binding points and to select C5aR antagonists based on the structure of the natural ligand.
Structural studies of histidine‐containing phosphocarrier protein from Enterococcus faecalis
Authors: Hahmann Marika , Maurer Till , Lorenz Michael , Hengstenberg Wolfgang , Glaser Steffen , Kalbitzer Hans Robert
Published: March 2003

Based on the complete sequential assignment of the 1H‐NMR spectrum by multidimensional NMR techniques the secondary structure and the local geometry of the active site of histidine‐containing phosphocarrier protein (HPr) from Enterococcus faecalis were elucidated. We present a comparative analysis of the active site in the seven known structures of HPr from different organisms determined by NMR or X‐ray crystallography. In catalysis, HPr is phosphorylated at the ring Nδ1 of His15. No general agreement exists in literature regarding the structure of the active‐centre loop. In the crystal structure of HPr from E. faecalis, a torsion strain of the backbone at position 16 was observed, which was assumed to be important to the catalytic mechanism. Coupling constants were determined in order to calculate φ angles to establish whether there are strained torsion angles in HPr from E. faecalis in the solution state. The evaluation of data obtained indicate a stable and well‐defined structure of HPr from E. faecalis, with an overall fold similar to that found in HPr from other bacteria. We find that in the active‐site region there are relatively large variations in local geometry between the evaluated structures. In HPr from E. faecalis, a particularly detailed view of the phosphate‐binding His15 and residues in close spatial proximity was obtained by determination of coupling constants obtained from the double‐quantum‐filtered COSY spectrum. Our data indicate that in aqueous solution, in the dominant conformational state there is no torsion strain of the backbone at position 16, as observed in the crystal state. The maximum population of a strained conformation in solution can be estimated to be smaller than 23 %. The analysis of the data suggests that the active‐centre loop is able to adopt different conformations in solution. A similar observation was made for HPr from E. faecalis phosphorylated at its regulatory site (Ser46). 31P‐NMR shows that phosphorylated HPr exists in two conformational substates with nearly equal populations.
Characterisation of peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A and its N‐glycans
Authors: Altmann Friedrich , Paschinger Katharine , Dalik Thomas , Vorauer Karola
Published: March 2003

Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two‐dimensional electrophoresis. Peaks corresponding to molecular masses of 54.2, 21.2 and 75.5 kDa were observed with matrix‐assisted laser‐desorption/ionization mass spectrometry. The N‐terminal sequences of the larger and the smaller chain were determined to be LASGYHSWAD and EPTPLHDFPP, respectively. Both polypeptides reacted with concanavalin A, indicating their glycoprotein nature.

Upon digestion of PNGase with pepsin, the N‐linked oligosaccharides were released with active PNGase and anlysed as their 2‐aminopyridine derivatives by two‐dimensional HPLC and by matrix‐assisted laser‐desorption mass spectrometry. The most abundant N‐glycan of the four species found exhibited the well known vacuole type structure, i.e. the pentasaccharide core with xylose and α1,3‐linked fucose. The other structures either had an additional mannose residue and/or lacked the fucose.

PNGase A was largely but not absolutely resistant to self‐deglycosylation. However, only at an extremely high enzyme/substrate ratio, N‐glycans released from PNGase A itself caused a detectable contamination of a PNGase digest of a glycopeptide.
Negative control of the Mig1p repressor by Snf1p‐dependent phosphorylation in the absence of glucose
Authors: Östling Jonas , Ronne Hans
Published: March 2003

Mig1p, a zinc‐finger protein that is related to the Krox/Egr, Wilms' tumor and Sp1 proteins, mediates glucose repression in the yeast Saccharomyces cerevisiae. Mig1p is inactive in the absence of glucose, and this inhibition is dependent on the Snf1p (Cat1p) protein kinase. The regulation is mediated by an internal part of Mig1p, and it can be transferred to a Mig1‐viral protein 16 (VP16) fusion protein that functions as an activator [Östling, J., Carlberg, M. & Ronne, H. (1996) Mol. Cell. Biol. 16, 753−761]. We have used Mig1‐VP16 to identify three target sites for phosphorylation that mediate Snf1p‐dependent inhibition of its activity in the absence of glucose. Two of the sites, Ser278 and Ser311, fit the consensus sequence for phosphorylation by the kinase Snf1p, as determined in vitro. However, a third phosphorylated site, Ser108, does not resemble a Snf1p site. We tested the effect of deleting residues 181−245, which contain two conserved alanine‐leucine‐serine motifs. We found that the deletion produces a partially constitutive activator, indicating that this region plays a general negative role in regulating Mig1p.
The accessible surface of the nicotinic acetylcholine receptor
Authors: Watty Anke , Weise Christoph , Dreger Mathias , Franke Peter , Hucho Ferdinand
Published: March 2003

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross‐linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand‐binding site, and cross‐links lysine residues located not more than 11 Å, the length of the cross‐linker, apart. Using this method, we identified a cross‐link located between βLys177 and βLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross‐linking also occurred between the vicinal lysine residues αLys76 and αLys77, indicating that these neighbouring lysine residues are not involved in a β‐sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues αLys76 and αLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202−8209] this might be due to their close proximity to the ligand binding site.
Secondary structure of the intact H+,K+‐ATPase and of its membrane‐embedded region
Authors: Raussens Vincent , de Jongh Harmen , Pézolet Michel , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

Models of P‐type ATPase predict that membrane‐embedded fragments represent about 20 % of the protein and adopt an all‐α‐helical structure. While this prediction was confirmed for the Ca2+‐ATPase [Corbalan‐Garcia, S., Teruel, J., Villalain, J. & Gomez‐Fernandez, J. (1994) Biochemistry 33, 8247−8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+‐ATPase [Vigneron, L., Ruysschaert, J.‐M. & Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17 685−17 696] and on the gastric H+,K+‐ATPase [Raussens, V., Ruysschaert, J.‐M. & Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262−270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane‐protected fragments of the H+,K+‐ATPase. Secondary structure of the intact and of the membrane‐protected segments was compared for oriented membrane films by attenuated total‐reflection Fourier‐transform infrared spectroscopy and by circular dichroism and for vesicles suspension by circular dichroism and Raman spectroscopy. All the spectroscopic data indicate that the protease‐resistant membrane‐bound residue of the H+,K+‐ATPase contains significant amount of β‐sheet structure, both on films and in membrane suspensions. Polarized attenuated total‐reflection infrared spectroscopy indicates that only the α‐helical content of protease‐resistant membrane‐bound residue of the H+,K+‐ATPase is oriented (parallel) with respect to the membrane normal. Raman spectroscopy reveals that Phe residues are preferentially removed by protease activity. Evaluation of the amount of removed Phe and Tyr residues places constraints on the model of membrane insertion of the H+,K+‐ATPase.
Comparative study of the catalytic domain of phosphorylating glyceraldehyde‐3‐phosphate dehydrogenases from bacteria and archaea via essential cysteine probes and site‐directed mutagenesis
Authors: Talfournier François , Colloc'h Nathalie , Mornon Jean‐Paul , Branlant Guy
Published: March 2003

Phosphorylating glyceraldehyde‐3‐phosphate dehydrogenase (GraP‐DH) catalyzes the oxidative phosphorylation of
D‐glyceraldehyde‐3‐phosphate to form 1.3‐diphosphoglycerate. The currently accepted mechanism involves an oxidoreduction step followed by a phosphorylation. Two essential aminoacids, Cys149 and His176 are involved in the chemical mechanism of bacterial and eukaryotic GraP‐DHs. Roles have been assigned to the His176 as (a) a chemical activator for enhancing the reactivity of Cys149, (b) a stabilizator of the tetrahedral transition states, and (c) a base catalyst facilitating hydride transfer towards NAD. In a previous study carried out on Escherichia coli GraP‐DH [Soukri, A., Mougin, A., Corbier, C., Wonacott, A. J., Branlant, C. & Branlant, G. (1989) Biochemistry 28, 2586−2592], the role of His176 as an activator of the reactivity of Cys149 was studied. Here, we further investigated the role of the His residue in the chemical mechanism of phosphorylating GraP‐DH from E. coli and Bacillus stearothermophilus. The chemical reactivity of Cys149 in the His176Asn mutant was reinvestigated. At neutral pH, its reactivity was shown to be at least as high as that observed in the Cys−/His+ ion pair present in the wild type. No pre‐steady state burst of NADH was found with the His176Asn mutant in contrast to what is observed for the wild type, and a primary isotope effect was observed when
D‐[1‐2H]glyceraldehyde‐3‐phosphate was used as the substrate. Therefore, the major role of the His176 in the catalytic mechanism under physiological conditions is not to activate the nucleophilicity of Cys149 but first to facilitate the hydride transfer. These results hypothesized that a phosphorylating GraP‐DH possessing a different protein environment competent to increase the nucleophilic character of the essential Cys residue and to favor the hydride transfer in place of His, could be enzymically efficient. This is most likely the case for archaeal Methanothermus fervidus GraP‐DH which shares less than 15 % amino‐acid identity with the bacterial or eukaryotic counterparts. No Cys−/His+ ion pair was detectable. Only one thiolate entity was observed with an apparent pKa of 6.2. This result was confirmed by the fact that none of the mutations of the five invariant His changed the catalytic efficiency.
How Drosophila species acquire cold tolerance
Authors: Ohtsu Takashi , Kimura Masahito T. , Katagiri Chihiro
Published: March 2003

Phospholipids of many cold‐tolerant organisms have been reported to contain more unsaturated fatty acids than cold‐susceptible organisms, a phenomenon known to maintain membrane fluidity at low temperature. However, we have obtained results to the contrary through a comparison of the membrane phospholipids of six temperate and subtropical species belonging to the Drosophila melanogaster species group. With enhancement of cold tolerance, the percentages of monoenoic acids increased but the percentages of dienoic acids decreased, that is, the number of double bonds in the phospholipid decreased without a marked variation in the percentages of unsaturated fatty acids. Concomitantly, the percentage of fatty acids containing 16 carbon atoms increased, while that of fatty acids with 18 carbon atoms decreased. Since phosphatidylethanolamine is a dominant phospholipid in Drosophila, these changes probably contribute to keeping the homeoviscosity of the cellular membranes in a manner different to that in phosphatidylcholine‐rich membranes, thereby increasing cold tolerance.
Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co‐mitogenic effects of ethanolamines and promotes cell survival
Authors: Malewicz Barbara , Mukherjee Jagat J. , Crilly Karan S. , Baumann Wolfgang J. , Kiss Zoltan
Published: March 2003

Ethanolamine (Etn), as well as its N‐methyl (MeEtn) and N,N‐dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum‐starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co‐mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn‐kinase‐mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P‐NMR analysis of the water‐soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum‐starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin‐induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50−200 μM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non‐phosphorylated ethanolamines are negative and positive regulators of insulin‐induced mitogenesis, respectively. After incubating the cells for 13 days in serum‐free medium in 96‐well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6−13 days, 0.1−1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.
Expression and characterization of three tomato 1‐aminocyclopropane‐1‐carboxylate oxidase cDNAs in yeast
Authors: Bidonde Sylvie , Ferrer Maria Angeles , Zegzouti Hicham , Ramassamy Sabine , Latché Alain , Pech Jean‐Claude , Hamilton Andrew J. , Grierson Donald , Bouzayen Mondher
Published: March 2003

Heterologous expression in yeast has previously shown that the tomato cDNA LE‐ACO1 encodes a functional 1‐aminocyclopropane‐1‐carboxylate (ACC) oxidase (ACO) protein [Hamilton, A. J., Bouzayen, M. & Grierson, D. (1991) Proc. Natl Acad. Sci. USA 88, 7434−7437]. In the present work, full‐length cDNAs encoding the two other members of the tomato ACO family (LE‐ACO2 and LE‐ACO3) were isolated and expressed in Saccharomyces cerevisiae. Analysis of the predicted amino acid sequences showed that the ACO1 and ACO3 proteins are highly similar (95 %) while ACO2 is more divergent (89 %). Yeast strains transformed with each of the three cDNAs were able to convert exogenous ACC to ethylene, the ACO1 strain exhibiting the highest activity in vivo and the ACO3 and ACO2 strains reaching 65 % and 45 % of ACO1 maximum activity, respectively. None of the ACO activities expressed in yeast required addition of ascorbate in vivo. ACO activities assayed in vitro revealed no significant differences between the three isoforms with regards to optimum temperature (29 °C), optimum pH (6.8−7.2), absolute dependence for ascorbate, Fe2+ and carbon dioxide, and inhibition by iron‐chelating agents (1,10‐phenanthroline and EDTA), Co2+ and free‐radical scavengers (n‐propyl gallate). However, differences were detected in the apparent Km values for ACC, the pI and the specific activity. The biochemical features that might explain the differences between the isoenzyme activities are discussed.
Bilitranslocase can exist in two metastable forms with different affinities for the substrates
Authors: Passamonti Sabina , Battiston Lucia , Sottocasa Gian Luigi
Published: March 2003

Bilitranslocase is an organic anion carrier involved in bilirubin and phthalein uptake by the liver. In rat liver plasma membranes, its function is assayed by recording the electrogenic sulfobromophthalein movement. This has been found to be inhibited by both cysteine‐specific and arginine‐specific reagents. Inhibition is both partial and it occurs to the same extent, i.e. approximately 50  %. The effects are not additive. Here we describe the mechanism underlying the above observations. It is concluded that bilitranslocase occurs in two possible states, featured by high and low affinity for the substrates (for sulfobromophthalein, Km = 5 μM and 37 μM, respectively). Cysteine‐ or arginine‐reactive reagents, by reacting selectively with the low‐affinity form, entrap it and shift the equilibrium between the two forms, so that, at completion, only the low‐affinity form is present. The substrate concentration in the standard transport assay is 39 μM, a value at which the modified low‐affinity form operates in the range of half‐maximal velocity. This explains both the apparent half‐inhibition measured after the chemical treatments and the lack of additivity. In addition, the substrates are shown to enhance the rate of conversion from the low‐affinity to the high‐affinity form of the translocator, thus favouring its high‐affinity form under physiological conditions.
Cholesterol, but not its esters, triggers programmed cell death in human erythroleukemia K562 cells
Authors: Maccarrone Mauro , Bellincampi Lorenza , Melino Gerry , Finazzi Agrò Alessandro
Published: March 2003

Cholesterol, its biosynthetic precursors and the cholesterol‐lowering drug compactin were able to inhibit the growth of human erythroleukemia K562 cells. Compactin, farnesyldiphosphate and cholesterol were cytotoxic by the induction of apoptosis (programmed cell death, PCD). Compactin doubled the number of apoptotic cells compared to control numbers, whereas farnesyldiphosphate and cholesterol led to a fivefold increase in PCD over the control levels. At variance with cholesterol, cholesterol esters did not affect K562 cell viability and apoptotic body formation, regardless of chain length and degree of saturation. Compactin and farnesyldiphosphate reduced the membrane cholesterol content, thus increasing membrane fluidity. Conversely, cholesterol treatment reduced the membrane fluidity by increasing cholesterol content in the lipid bilayer. Unlike farnesyldiphosphate, the other cholesterol precursors and cholesterol esters were ineffective in increasing the cholesterol content and, thereby, the fluidity of cell membranes. Compactin and cholesterol precursors, apart from farnesyldiphosphate, did not affect the amount of the farnesylated proteins Ras and lamin B in the cytosolic and the membrane fractions of K562 cell extracts, whereas farnesyldiphosphate reduced the content of both proteins in both fractions. The level of lamin B in K562 cytosol and membranes was also reduced by cholesterol treatment, which did not significantly affect the amount of Ras. These findings highlight the role of cholesterol in promoting PCD.
Solution structures of the fibronectin‐like Leishmania gp63 SRYD‐containing sequence in the free and antibody‐bound states
Authors: Petit Marie‐Christine , Orlewski Piotr , Tsikaris Vassilios , Sakarellos‐Daitsiotis Maria , Sakarellos Constantinos , Tzinia Athina , Konidou Georgia , Soteriadou Ketty P. , Marraud Michel , Cung Manh Thong
Published: March 2003

The anti‐SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250−257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD‐containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD‐containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two‐dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody‐bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton‐distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted γ‐turn was found, stabilized by the backbone−backbone D255‐NH to R253‐CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody‐bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL−receptor interaction is discussed.
Separation of horse dander allergen proteins by two‐dimensional electrophoresis
Authors: Bulone Vincent , Krogstad‐Johnsen Tonje , Smestad‐Paulsen Berit
Published: March 2003

The aim of this work was to identify which proteins in horse dander extracts are allergens and to characterise them. Two‐dimensional PAGE showed that horse dander preparations are composed of up to 50 proteins, all having acidic isoelectric points in the pH range 3−4.5. Immunoblots of two‐dimensional PAGE were used to compare the reactivity of the proteins with IgE from 23 allergic patients. Patient sera were divided into two main groups recognising either allergens of 18.5 kDa or proteins of 27−29 and 31 kDa. The proteins of 27−29 kDa and 31 kDa were all N‐glycosylated and their glycan chains seem to play a role in the binding of IgE from allergic patients. The sugar composition of their carbohydrate moiety was determined and lectin‐binding experiments indicated presence of terminal sialic acid linked α‐(2←6) to galactose, galactose linked β‐(1←4) to N‐acetylglucosamine, and possibly presence of sialic acid linked α‐(2←3) to galactose. The 27−29‐kDa glycoproteins had heterogeneous isoelectric points, most probably due to different degrees of sialylation in their oligosaccharide chains. The two 18.5‐kDa allergens exhibited slightly different isoelectric points and their N‐terminal sequences were identical, showing that they most likely were isoforms of the same protein. Sequence analyses revealed that their N‐terminal sequences are similar to proteins belonging to the lipocalin family. We named the two 18.5‐kDa proteins Equ c 2.0101 and Equ c 2.0102, according to International Allergen Nomenclature recommendations [King, T. P., Hoffman, D., Løwenstein, H., Marsh, D. G., Platts‐Mills, T. A. E. & Thomas, W. (1995) J. Allergy Clin. Immunol. 96, 5−14]. The N‐terminal of the allergens of 27−29 kDa were blocked and their sequences were not determined. Their amino acid compositions were determined and comparison with acidic mammalian proteins in the Swiss‐Prot database revealed high scores with lipocalin proteins. This suggests that the glycosylated horse dander allergens belong to the lipocalin family, like Equ c 2.0101 and Equ c 2.0102.
Solution structure of the antimicrobial peptide ranalexin and a study of its interaction with perdeuterated dodecylphosphocholine micelles
Authors: Vignal Emmanuel , Chavanieu Alain , Roch Philippe , Chiche Laurent , Grassy Gérard , Calas Bernard , Aumelas André
Published: March 2003

Ranalexin, a 20‐residue peptide isolated from the skin of the bullfrog Rana catesbeiana displays antimicrobial activity. This peptide contains two cysteine residues in positions 14 and 20 linked by a disulphide bridge. Ranalexin was chemically synthesised and close antimicrobial activities were measured for the reduced and oxidised forms. The solution structure of ranalexin was determined by using circular dichroism, proton NMR spectroscopy and molecular modelling techniques. The reduced and oxidised forms of ranalexin are mainly unstructured in water but display an α‐helical structure spanning residues 8−15 and 8−17, respectively, in a trifluoroethanol/water mixture (3 : 7, by vol.). Ranalexin was found to interact with micelles of dodecylphosphocholine and to adopt a similar helical structure. Moreover, slow‐exchanging amide protons located on the same side of the helix suggest that the hydrophobic face of the helix lies on the micelle surface. Hydrophobic residues of the poorly structured N‐terminal part which are important for the biological activity are also involved in the interaction with micelles. Taken together, the results suggest that the disulphide bond does not strongly affect either the conformation or the antimicrobial activity of ranalexin.
Role of sialosyl Lewisa in adhesion of colon cancer cells
Authors: Kłopocki Arkadiusz G. , Laskowska Anna , Antoniewicz‐Papis Jolanta , Duk Maria , Lisowska Elwira , Ugorski Maciej
Published: March 2003

To study whether the adhesion of colon cancer cells to E‐selectin can be directly affected by changes in the expression level of sialosyl Lea antigen we created a specific loss‐of‐function phenotype. A stable subclone (CX‐1.1) with high expression of sialosyl Lea structure, obtained from a heterogenous population of colon carcinoma CX‐1 cells, was transfected with an expression vector containing a fragment of cDNA for α1,3/4‐fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Lea antigen and lacked the α1,3/4‐fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Lea carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E‐selectin. To evaluate which cellular glycoconjugates carry sialosyl Lea antigen, glycoproteins as well as glycolipids of CX‐1.1 cells were analysed for the expression of this structure. Anti‐sialosyl Lea antibodies detected multiple glycoprotein bands with apparent molecular masses of 65−280 kDa on western blots, and an intense band representing sialosyl Lea‐ganglioside on a thin‐layer chromatogram. Using O‐sialoglycoprotease from Pasteurella haemolytica and an alkaline β‐elimination procedure, it was shown that protein‐linked sialosyl Lea structures are carried mostly by mucin‐type glycoproteins. However, treatment of CX‐1.1 cells with O‐sialoglycoprotease did not decrease either their binding to E‐selectin‐expressing Chinese hamster ovary cells, or binding of anti‐sialosyl Lea antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Lea‐ganglioside, which was inaccessible for the antibody and E‐selectin in untreated cells. This hypothesis was confirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O‐sialoglycoprotease‐treated CX‐1.1 cells, comparing to untreated cells. We propose that glycoproteins as well as gangliosides carrying sialosyl Lea structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to E‐selectin.
Monitoring of RNA polymerase−DNA UP element interaction by a fluorescent probe conjugated to α subunit
Authors: Ozoline Olga N. , Fujita Nobuyuki , Murakami Katsuhiko , Ishihama Akira
Published: March 2003

The carboxy‐terminal domain (CTD) of Escherichia coli RNA polymerase α subunit was specifically modified by a reporter label, fluorescein mercuric acetate (FMMA), conjugated to Cys269 on the surface of UP element recognition helix. The modified enzyme was used to investigate RNA polymerase interaction with different promoters, either with or without an UP element. In a single‐round transcription assay, the activity of modified RNA polymerase was found to decrease as measured with rrnBP1, trpP and lacP2 promoters but not with many other promoters including mutant rrnBP1 without the UP element, supporting the idea that Cys269 or the domain including Cys269 is involved in UP element recognition. Both trpP and lacP2 have sequence similarity to the rrnBP1 UP element. The chemical modification of RNA polymerase, however, did not affect an apparent equilibrium dissociation constant with rrnBP1, as measured by gel‐retardation assays, indicating that the DNA‐binding ability is retained even after FMMA conjugation. Interaction with the rrnBP1 UP element led to substantial alterations in the spectral parameters of the reporter label, which are different from those induced by complex formation with promoters without UP elements. A pronounced spectral blue shift suggests that the labeled surface of αCTD closely approaches the charged UP DNA helix. These observations imply that the fluorescent labeling at Cys269 can be used as a good tool for monitoring the presence or absence of an UP element in a given promoter. Spectral parameters of the label displayed the spectral blue shift when the modified RNA polymerase interacted with trpP, supporting the prediction that this promoter carries an rrnBP1‐type UP element.
Pigment‐binding properties of the recombinant photosystem II subunit CP26 reconstituted in vitro
Authors: Ros Francesca , Bassi Roberto , Paulsen Harald
Published: March 2003

CP26 is the most recently described antenna protein in higher plants which has been reported to be involved in xanthophyll‐dependent regulation of the light‐harvesting function but is largely unknown due to the difficulties of purification. In this study we have overexpressed in Escherichia coli the Lhcb5 gene product and reconstituted CP26 in vitro by refolding the recombinant protein in the presence of chlorophyll a, chlorophyll b and xanthophylls. The resulting pigment‐protein complex is stable enough to be isolated by partially denaturing gel electrophoresis. Reconstitution and isolation conditions for CP26 are similar to those used for other chlorophyll a/b complexes like the major light‐harvesting complex of photosystem II (LHCII) and CP29; however, CP26 differs with regard to its lower specificity in carotenoid binding. Most significantly, rather stable recombinant CP26 can be reconstituted containing violaxanthin as the only carotenoid. This enhanced plasticity with respect to carotenoid binding is consistent with CP26 being the major binding protein of violaxanthin involved in the xanthophyll cycle. The availability of recombinant CP26 opens the way to a better characterisation of this pigment‐protein complex with regard to its biochemistry and its physiological functions.
Purification and characterization of dimethylamine : 5‐hydroxybenzimidazolyl‐cobamide methyltransferase from Methanosarcina barkeri Fusaro
Authors: Wassenaar Roel W. , Keltjens Jan T. , Van Der Drift Chris , Vogels Godfried D.
Published: March 2003

Dimethylamine : 5‐hydroxybenzimidazolylcobamide methyltransferase (DMA‐MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine :coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2‐mercaptoethanesulfonate (coenzyme M) to monomethylamine and methyl‐coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA‐MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50‐kDa subunits. A corrinoid content of 0.9 ± 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as‐isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase‐activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.
Molecular cloning, gene structure and expression profile of mouse C1 inhibitor
Authors: Lener Matteo , Vinci Giovanna , Duponchel Christiane , Meo Tommaso , Tosi Mario
Published: March 2003

The gene encoding C1 inhibitor, the major control element of activation of the classical pathway of complement and a major inhibitor of several plasma serine proteases, has been studied only in man, where deficiency of C1 inhibitor results in the dominantly transmitted disease hereditary angioedema. Full‐length mouse C1 inhibitor cDNA and genomic clones were isolated and characterized as a first step towards the complete characterization of the pattern of C1 inhibitor expression and the production of an animal model of C1 inhibitor deficiency. Restriction‐enzyme and sequence analyses of a full‐length genomic clone demonstrated that the mouse gene has the same structure as the human homologue, but differs in size (9 kb versus 17 kb), mostly due to the presence of repetitive Alu elements in the human gene. Sequence comparisons in the promoter region indicate important similarities, i.e. the absence of a TATA box, the presence of an initiator sequence encompassing the transcription‐start site and of a γ‐interferon‐activated sequence (GAS) element at position −124 of the human sequence. A stretch of about 100 nucleotides in intron 1 reveals an unusually high degree of conservation for non‐coding sequences and contains non‐canonical but conserved tandemly arranged GAS elements at positions 369 and 388 of the human sequence. This finding supports the conclusions of functional studies on the human C1INH gene indicating a role of this region in modulation of transcription by interferons. The profile of C1 inhibitor expression in mouse liver, lung, heart, kidney, spleen and brain was determined by quantitative northern blot analysis.
Stoichiometry of coupling of rhodamine 123 transport to ATP hydrolysis by P‐glycoprotein
Authors: Shapiro Adam B. , Ling Victor
Published: March 2003

In order to describe the transport mechanism of P‐glycoprotein, it is essential to know the coupling ratio, i.e. the moles substrate transported/mole ATP hydrolyzed. P‐glycoprotein couples ATP hydrolysis at two ATP‐binding sites to transport of a wide variety of neutral or cationic lipophilic compounds. Previously published coupling ratios have fallen within the range 0.02−0.8 mol substrate transported/mol ATP hydrolyzed. We studied the energetics of transport by P‐glycoprotein, performing quantitative measurements of the rates of ATP hydrolysis and transport of rhodamine 123 by P‐glycoprotein, using isolated P‐glycoprotein‐rich plasma membrane vesicles. The continuous fluorescence‐based assay of rhodamine 123 transport allowed accurate measurement of initial transport rates. Since we measured uptake of rhodamine 123 into the vesicles as a loss of fluorescence, we avoided the problem of high background due to substrate binding to the membranes. The coupling ratio of the transport reaction increased as the rhodamine 123 concentration increased, showing that the basal ATPase activity of P‐glycoprotein was progressively recruited for rhodamine 123 transport. Both of the previously identified transport sites of P‐glycoprotein [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130−137] were involved in transport of saturating concentrations of rhodamine 123. At saturating rhodamine 123 and 0.3 mM ATP, the coupling ratio was 0.83, suggesting a mechanistic coupling ratio of 1. Interestingly, the coupling ratio decreased as the ATP concentration increased so that, at 1.5 mM, close to the cytoplasmic concentration of ATP, the coupling ratio was 0.57. The physiological significance of this effect is not yet understood.