bluuebunny/crossref_metadata_2025 huggingface dataset:
Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa
Authors: Hill Justine M. , Atkins Annette R. , Loughnan Marion L. , Jones Alun , Adams Denise A. , Martin Rod C. , Lewis Richard J. , Craik David J. , Alewood Paul F.
Published: March 2003

A novel conotoxin belonging to the ‘four‐loop’ structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical‐directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino‐acid residues and contains three disulfide bonds. The amino‐acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4‐transl‐hydroxyproline. Two under‐hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four‐loop structural framework common to many peptides from Conus venoms including the ω‐, δ‐ and κ‐conotoxins. However, TVIIA displays little sequence homology with these well‐characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four‐loop conotoxins.
Reconstitution of ethanolic fermentation in permeabilized spheroplasts of wild‐type and trehalose‐6‐phosphate synthase mutants of the yeast Saccharomyces cerevisiae
Authors: Noubhani Abdelmajid , Bunoust Odile , Rigoulet Michel , Thevelein Johan M.
Published: March 2003

In the yeast Saccharomyces cerevisiae, TPS1‐encoded trehalose‐6‐phosphate synthase (TPS) exerts an essential control on the influx of glucose into glycolysis, presumably by restricting hexokinase activity. Deletion of TPS1 results in severe hyperaccumulation of sugar phosphates and near absence of ethanol formation. To investigate whether trehalose 6‐phosphate (Tre6P) is the sole mediator of hexokinase inhibition, we have reconstituted ethanolic fermentation from glucose in permeabilized spheroplasts of the wild‐type, tps1Δ and tps2Δ (Tre6P phosphatase) strains. For the tps1Δ strain, ethanol production was significantly lower and was associated with hyperaccumulation of Glu6P and Fru6P. A tps2Δ strain shows reduced accumulation of Glu6P and Fru6P both in intact cells and in permeabilized spheroplasts. These results are not consistent with Tre6P being the sole mediator of hexokinase inhibition. Reconstitution of ethanolic fermentation in permeabilized spheroplasts with glycolytic intermediates indicates additional target site(s) for the Tps1 control. Addition of Tre6P partially shifts the ethanol production rate and the metabolite pattern in permeabilized tps1Δ spheroplasts to those of the wild‐type strain, but only with glucose as substrate. This is observed at a very high ratio of glucose to Tre6P. Inhibition of hexokinase activity by Tre6P is less efficiently counteracted by glucose in permeabilized spheroplasts compared to cell extracts, and this effect is largely abolished by deletion of TPS2 but not TPS1. In permeabilized spheroplasts, hexokinase activity is significantly lower in a tps2Δ strain compared to a wild‐type strain and this difference is strongly reduced by additional deletion of TPS1. These results indicate that Tps1‐mediated protein–protein interactions are important for control of glucose influx into yeast glycolysis, that Tre6P inhibition of hexokinase might not be competitive with respect to glucose in vivo and that also Tps2 appears to play a role in the control of hexokinase activity.
Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket
Authors: Couture Manon , Das Tapan Kanti , Savard Pierre‐Yves , Ouellet Yannick , Wittenberg Jonathan B , Wittenberg Beatrice A , Rousseau Denis L , Guertin Michel
Published: March 2003

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe‐protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s−1, is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6‐coordinate and low‐spin in both ferric and ferrous forms in the pH range 5.5–10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino‐acid sequence comparison, is involved in the formation of the ferric and ferrous 6‐coordinate low‐spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5‐coordinate and high‐spin and the ferric form contains a 6‐coordinate high‐spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.
Differential degradation of Escherichia coliσ32 and Bradyrhizobium japonicum RpoH factors by the FtsH protease
Authors: Urech Carmen , Koby Simi , Oppenheim Amos B. , Münchbach Martin , Hennecke Hauke , Narberhaus Franz
Published: March 2003

The Escherichia coli heat shock sigma factor σ32 (RpoH) is rapidly degraded under non‐stress conditions. The integrity of the DnaK chaperone machinery and the ATP‐dependent FtsH protease are required for σ32 proteolysis in vivo. Bradyrhizobium japonicum expresses three σ32‐type transcription factors, RpoH1, RpoH2, and RpoH3, which are functional in E. coli. We compared the stability of these sigma factors with E. coliσ32 stability. In E. coli C600 (wild‐type), the half‐lives of σ32, RpoH1, RpoH2 and RpoH3 were 30 s, 7 min, 4 min and 4 min, respectively. The first three proteins were stabilized in ftsH mutant backgrounds, indicating that they are degraded by FtsH in the wild‐type. Proteolysis of RpoH3 was FtsH‐independent because this sigma factor was not stabilized in ftsH mutants. Interestingly, in a purified in vitro system, all four RpoH proteins were degraded by FtsH, indicating that in vivo protein degradation depends on additional cellular factors. Rationally designed point mutations of σ32 and RpoH1 suggested that the highly conserved RpoH box does not play a major role in conferring stability to RpoH factors. Presumably, several regions distributed along the primary sequence of the sigma factor are important for FtsH‐mediated proteolysis. Finally, we provide evidence that proteolysis of RpoH factors in vivo depends on the DnaK machinery, irrespective of the protease involved.
Towards the selection of phosphorothioate aptamers
Authors: Andreola Marie‐Line , Calmels Christina , Michel Justine , Toulmé Jean‐Jacques , Litvak Simon
Published: March 2003

The high affinity of a given nucleic acid for a protein ligand can be used to isolate specific inhibitors of enzymes involved in pathological situations. The latter property is the basis of the SELEX (systematic evolution of ligands by exponential enrichment) technique. Recently, several potent nucleic acids inhibitors of HIV‐1 replication have been isolated using the SELEX approach. However, phosphodiester oligodeoxynucleotides (PO‐ODNs) were not used as antiviral agents because of their sensitivity to nucleases. Our goal in this work was to explore the possibility of selecting, from a fully substituted phosphorothioate library, oligonucleotides having both a strong affinity for HIV‐1 reverse transcriptase (RT) and nuclease resistance. HIV‐1 RT initiates in vivo reverse transcription from the 3′ end of a host tRNALys. Although phosphorothioate ODNs (PS‐ODNs) have been claimed to bind unspecifically to proteins, we have shown previously that an ODN corresponding to the acceptor stem of tRNALys was able to inhibit specifically HIV‐1 replication in HIV‐1 infected cells, without showing cytotoxicity up to 10 µm. As the SELEX strategy requires ‘in vitro’ transcription and reverse transcription of the selected DNA, we have assayed the available PS precursors as a model system by using PS‐dNTPs and rNTPs. We have also developed an experimental procedure to optimize the incorporation of four PS‐dNTPs during the PCR step of the SELEX approach. In the course of this work, we have showed that the PS‐dGTP is a strong inhibitor of thermostable DNA polymerases as well as of HIV‐1 RT.
Increased formation of reactive oxygen species due to glucose depletion in primary cultures of rat thymocytes inhibits proliferation
Authors: Aulwurm Uwe R. , Brand Karl A.
Published: March 2003

Glycolytic glucose degradation to lactate is the only way of energy production in mammalian cells that does not use oxygen, and is thus a means of reducing oxidative stress. The aim of this work was to study the effect on proliferation of progressive glucose depletion during the cell cycle of stimulated cultured rat thymocytes. It has been shown that under conditions of limited glucose supply, these cells increase the generation of reactive oxygen species. Because of the sensitivity of the stimulating transcription factor Sp1 to redox changes with respect to its DNA binding, gene expression of glycolytic enzymes is markedly suppressed. As a consequence, cell division does not occur in glucose depleted thymocytes.
The plant aspartic proteinase‐specific polypeptide insert is not directly related to the activity of oryzasin 1
Authors: Asakura Tomiko , Matsumoto Ichiro , Funaki Junko , Arai Soichi , Abe Keiko
Published: March 2003

Many plant aspartic proteinases (APs) are different from animal and microbial APs in that they contain a polypeptide insert, ≈ 100 amino acids in length, in the C‐terminal region. To interpret the significance of this insert, we constructed an expression system for rice AP oryzasin 1 by linking a pro‐oryzasin 1 downstream of glutathione S‐transferase (GST). GST–proOS1 expressed the highest degree of hemoglobin‐hydrolytic activity when treated at pH 3.3 and incubated for 24 h at room temperature. We carried out a similar experiment using an insert‐lacking proOS1 mutant, GST–ΔproOS1, as the fusion protein, and found it to show similar activity. This result indicates that the insert is not involved in the production of AP activity. We then investigated the autolysis of the two proteins by Western blot analysis. GST–proOS1 was autolyzed into 67‐ and 64‐kDa fragments, while GST–ΔproOS1 autolyzed to 54‐ and 52‐kDa products. GST–ΔproOS1 clearly produced two molecular species early in the autolytic process, and not later than 3 h from the start, but no such clear result was observed in the case of GST–proOS1. This suggests that, although the presence of the plant AP‐specific insert does not influence the enzyme activity by itself, it apparently has an effect on the autolysis of OS1.
Polypeptide chain composition diversity of hexagonal‐bilayer haemoglobins within a single family of annelids, the Alvinellidae
Authors: Zal Franck , Green Brian N. , Martineu Pascale , Lallier François H. , Toulmond André , Vinogradov Serge N. , Childress James J.
Published: March 2003

Following previous analysis of the structure of Alvinella pompejana heaxagonal‐bilayer haemoglobin (HBL Hb) [1], we report in this paper the structure of three other HBL Hbs belonging to Alvinella caudata, Paralvinella grasslei and Paralvinella palmiformis, members of the Alvinellidae, annelid family strictly endemic to deep‐sea hydrothermal vents located on the ridge crests in the Pacific ocean. The multi‐angle laser light scattering (MALLS) and fast protein liquid chromatography (FPLC) analysis revealed a broad range of molecular masses for the extracellular Hb molecules, 3517 ± 14 kDa (A. caudata), 3822 ± 28 kDa (P. grasslei) and 3750 ± 150 kDa (P. palmiformis). Native and derivative Hbs (reduced, carbamidomethylated and deglycosylated) were analysed by electrospray ionization mass spectroscopy (ESI‐MS) and the data was processed by the maximum entropy deconvolution system (MaxEnt). The most important difference between alvinellid HBL Hbs was the variation in their composition, from two to four monomeric globin chains, and from one to four linker chains. Therefore, despite the fact that all these species belong to a single family, notable differences in the polypeptide chain composition of their HBL Hbs were observed, probably accounting for their different functional properties as previously reported by this group Toulmond, A., El Idrissi Slitine, F., De Frescheville, J. & Jouin, C. (1990) Biol. Bull.179, 366–373.
The amino‐acid sequence of the abalone (Haliotis laevigata) nacre protein perlucin
Authors: Mann Karlheinz , Weiss Ingrid M. , André Sabine , Gabius Hans‐Joachim , Fritz Monika
Published: March 2003

Perlucin isolated from abalone nacre consists of 155 amino acids including a glycosylated asparagine. The sequence of the first 130 amino acids shows a high similarity to the C‐type carbohydrate‐recognition domains of asialoglycoprotein receptors and other members of the group of C‐type lectins but also a weaker similarity to related proteins without carbohydrate‐binding activity. This C‐type module is followed by a short C‐terminal domain containing two almost identical sequence repeats with a length of 10 amino acids. Solid phase assays show a divalent metal ion‐dependent binding of perlucin to (neo)glycoproteins containing d‐galactose or d‐mannose/d‐glucose indicating that perlucin is a functional C‐type lectin with broad carbohydrate‐binding specificity. Our results also indicate that it may be difficult to predict carbohydrate‐binding specificity and the occurrence of alternative binding configurations by amino‐acid sequence comparisons and homology modeling.
The light‐harvesting antenna of brown algae
Authors: De Martino Alessandra , Douady Dominique , Quinet‐Szely Michelle , Rousseau Bernard , Crépineau Florent , Apt Kirk , Caron Lise
Published: March 2003

Accessory light‐harvesting complexes (LHCFs) were isolated from the brown alga Laminaria saccharina. Complexes specifically associated with photosystem I or II are identical with each other with respect to molecular mass, isoelectric point and behavior on anion‐exchange chromatography or non‐denaturing isoelectric focusing. The purified complexes also have similar pigment composition and spectroscopic properties. It is concluded that LHC antennae associated with photosystem I or II cannot be distinguished biochemically. After screening of genomic and cDNA libraries produced from L. saccharina sporophytes, six lhcf genes were isolated. Sequence analysis of these lhcf genes showed a high level of homology between the encoded polypeptides. Comparisons with coding sequences of lhcf genes from Macrocystis pyrifera and expressed sequence tags from Laminaria digitata (two other Laminariales) indicated that these proteins are probably very similar in all brown algae. Another feature common to the lhcf genes characterized was the presence of an intron in the coding region corresponding to the plastid‐targeting presequence. The sequence similarity extended to the 5′ and 3′ UTRs of several genes. In spite of the common origin of the chloroplasts, no light‐regulating elements involved in the expression of the genes encoding the higher‐plant light‐harvesting proteins has been found in the UTRs.
Comparative analyses of secondary gene products of 3‐deoxy‐D‐manno‐oct‐2‐ulosonic acid transferases from Chlamydiaceae in Escherichia coli K‐12
Authors: Brabetz Werner , Lindner Buko , Brade Helmut
Published: March 2003

The waaA gene encoding the essential, lipopolysaccharide (LPS)‐specific 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K‐12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K‐12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure α‐Kdo‐(2→8)‐[α‐Kdo‐(2→4)]‐α‐Kdo‐(2→4)‐α‐Kdo.
Purification, characterization and molecular modelling of two toxin‐like proteins from the Androctonus australis Hector venom
Authors: Srairi‐Abid Najet , Mansuelle Pascal , Mejri Thouraya , Karoui Habib , Rochat Hervé , Sampieri François , El Ayeb Mohamed
Published: March 2003

Two toxin‐like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross‐reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins.

In order to study structure‐function relationships, their complete amino‐acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three‐dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion α‐toxins specific to voltage dependent Na+ channels. The models showed also that amino‐acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.
The unusual dodecameric ferritin from Listeria innocua dissociates below pH 2.0
Authors: Chiaraluce Roberta , Consalvi Valerio , Cavallo Stefano , Ilari Andrea , Stefanini Simonetta , Chiancone Emilia
Published: March 2003

The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size‐exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values ≥ 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three‐dimensional structure.
Glycosylation sites in the atrial natriuretic peptide receptor
Authors: Miyagi Masaru , Zhang Xiaolun , Misono Kunio S.
Published: March 2003

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR‐ECD) from rat expressed in COS‐1 cells. HPLC/MS analysis of a tryptic digest of NPR‐ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn‐linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high‐mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high‐mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR‐ECD followed by binding assay. NPR‐ECD deglycosylated with endoglycosidase F2 and endoglycosidase H retained ANP‐binding activity and showed an affinity for ANP similar to that of untreated NPR‐ECD. Endoglycosidase treatment of the full‐length ANP receptor expressed in COS‐1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.
Leptin stimulates uncoupling protein‐2 mRNA expression and Krebs cycle activity and inhibits lipid synthesis in isolated rat white adipocytes
Authors: Ceddia Rolando B. , William William N. , Lima Fabio B. , Flandin Pierre , Curi Rui , Giacobino Jean‐Paul
Published: March 2003

The treatment of rats and mice with leptin causes dramatic body fat reduction and in some cases even disappearance of fat tissue. Here, we report the effects of leptin (10 and 100 ng·mL−1) on isolated rat adipocytes maintained for 15 h in culture. Leptin decreased the incorporation of acetate into total lipids by 30%. A reduction in this incorporation (42%) was still observed after the leptin‐cultivated adipocytes were exposed to a supra‐physiological insulin concentration (10 000 µU·mL−1). On the other hand, leptin increased acetate degradation by 69% and the maximal activity of citrate synthase by 50% in isolated adipocytes. It also increased oleate degradation by 35 and 50% at concentrations of 10 and 100 ng·mL−1, respectively. Eventually, leptin upregulated the uncoupling protein‐2 (UCP2) mRNA level by 63% and had no effect on uncoupling protein‐3 (UCP3) mRNA in isolated adipocytes. The upregulation of UCP2 mRNA might have contributed to the stimulation of acetate and fatty acid degradation by leptin. The peripheral effects of leptin observed in this study are in line with the general energy dissipating role postulated for this hormone and for UCP2. They suggest mechanisms by which adipocytes regulate their fat content by an autocrine pathway without the participation of the central nervous system.
Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101)
Authors: Walsh Mark E. , Kyritsis Panos , Eady Nigel A. J. , Hill H. Allen O. , Wong Luet‐Lok
Published: March 2003

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active‐site polarity and volume were probed by combinations of active‐site mutations. Increasing the active‐site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side‐chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side‐chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W–V247L (80 min−1 or 2.5 × the activity of the wild‐type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side‐chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active‐site polarity and compromises were necessary to achieve reasonable values for both.
Gating of inward‐rectifier K+ channels by intracellular pH
Authors: Schulte Uwe , Fakler Bernd
Published: March 2003

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH‐gating involves structural rearrangements in cytoplasmic domains and the P‐loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a ‘pH sensor’. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N‐ and C‐termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions.

This article will focus on the mechanism of pH‐gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.
Characterization of porin isoforms expressed in tumor cells
Authors: Shinohara Yasuo , Ishida Taro , Hino Mami , Yamazaki Naoshi , Baba Yoshinobu , Terada Hiroshi
Published: March 2003

Mitochondria from malignant tumor cell lines show a higher capability for hexokinase binding than those from normal liver . To explore possible differences in hexokinase binding sites of mitochondria between tumor cells and normal liver, we characterized porin isoforms expressed in tumor cells. Cloning experiments on the three porin isoforms, VDAC1, VDAC2 and VDAC3 from malignant tumor cell line AH130 clearly showed that their primary structures were completely identical to those of the corresponding VDACs of normal liver cells. Possible expression of the fourth porin isoform in AH130 cells was excluded by degenerate primer‐based RT‐PCR. However, the transcript levels of the three VDAC isoforms in AH130 cells were significantly higher than those in normal liver. These results suggest that the high hexokinase‐binding capability of malignant tumor cell mitochondria was not due to any structural difference, but due to a quantitative difference in binding sites.
Preferential interaction of α‐tocopherol with phosphatidylcholines in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine
Authors: Wang Xiaoyuan , Quinn Peter J.
Published: March 2003

The effect of α‐tocopherol on the structure and phase behaviour of mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine has been examined by synchrotron X‐ray diffraction and freeze‐fracture electron microscopy. Equimolar mixtures of fully saturated derivatives of phospholipids that show gel phase immiscibility were examined including dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoethanolamine and distearoylglycerophosphocholine/dilauroylglycerophosphoethanolamine. Analysis of the X‐ray scattering intensities recorded at wide angles during heating scans of mixed aqueous dispersions containing 2.5 or 5 mol% α‐tocopherol showed that α‐tocopherol disordered the acyl chain packing of the phosphatidylcholine to a greater extent than the phosphatidylethanolamine component of the mixture. This suggested that α‐tocopherol preferentially interacts with phosphatidylcholine rather than phosphatidylethanolamine, irrespective of whether this was the high or low melting point component of the mixture. The presence of 20 mol% α‐tocopherol in either phospholipid mixture prevented gel phase separation during the prior cooling scan and no conclusions could be drawn as to the distribution of α‐tocopherol in these mixtures.
Dissociation and unfolding of cold‐active alkaline phosphatase from Atlantic cod in the presence of guanidinium chloride
Authors: Ásgeirsson Bjarni , Hauksson Jónas B. , Gunnarsson Gudmundur H.
Published: March 2003

Cold‐adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold‐active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size‐exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three‐state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two‐state model). The ΔG for dimer dissociation of cod AP was 8.3 kcal·mol−1, and the monomer stabilization free energy was 2.2 kcal·mol−1, giving a total of 12.7 kcal·mol−1, whereas the total free energy of calf intestinal AP was 17.3 kcal·mol−1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate‐limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.
Prostromelysin‐1 (proMMP‐3) stimulates plasminogen activation by tissue‐type plasminogen activator
Authors: Arza Begoña , Hoylaerts Marc F. , Félez Jordi , Collen Désiré , Lijnen H. Roger
Published: March 2003

Matrix metalloproteinase‐3 (MMP‐3 or stromelysin‐1) specifically binds to tissue‐type plasminogen activator (t‐PA), without however, hydrolyzing the protein. Binding affinity to proMMP‐3 is similar to single chain t‐PA, two chain t‐PA and active site mutagenized t‐PA (Ka of 6.3 × 106 to 8.0 × 106 m−1), but is reduced for t‐PA lacking the finger and growth factor domains (Ka of 2.0 × 106 m−1). Activation of native Glu‐plasminogen by t‐PA in the presence of proMMP‐3 obeys Michaelis–Menten kinetics; at saturating concentrations of proMMP‐3, the catalytic efficiency of two chain t‐PA is enhanced 20‐fold (kcat/Km of 7.9 × 10−3 vs. 4.1 × 10−4 µm−1·s−1). This is mainly the result of an enhanced affinity of t‐PA for its substrate (Km of 1.6 µm vs. 89 µm in the absence of proMMP‐3), whereas the kcat is less affected (kcat of 1.3 × 10−2 vs. 3.6 × 10−2 s−1). Activation of Lys‐plasminogen by two chain t‐PA is stimulated about 13‐fold at a saturating concentration of proMMP‐3, whereas that of miniplasminogen is virtually unaffected (1.4‐fold). Plasminogen activation by single chain t‐PA is stimulated about ninefold by proMMP‐3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys‐plasminogen to proMMP‐3 with 18‐fold higher affinity (Ka of 22 × 106 m−1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 × 106m−1) as compared to Glu‐plasminogen (Ka of 1.2 × 106m−1). Plasminogen and t‐PA appear to bind to different sites on proMMP‐3. These data are compatible with a model in which both plasminogen and t‐PA bind to proMMP‐3, resulting in a cyclic ternary complex in which t‐PA has an enhanced affinity for plasminogen, which may be in a Lys‐plasminogen‐like conformation. Maximal binding and stimulation require the N‐terminal finger and growth factor domains of t‐PA and the N‐terminal kringle domains of plasminogen.
Temporal secretion of a multicellulolytic system in Myxobacter sp. AL‐1
Authors: Avitia C. I. , Castellanos‐Juárez F. X. , Sánchez E. , Téllez‐Valencia A. , Fajardo‐Cavazos P. , Nicholson W. L. , Pedraza‐Reyes M.
Published: March 2003

The Gram‐negative soil micro‐organism Myxobacter sp. AL‐1 possesses at least five extracellular cellulases, the production of which is regulated by the growth cycle. We cloned the complete gene for one of these cellulases, termed cel9, which encoded a 67‐kDa modular family 9 endoglycohydrolase, which was produced during the stationary phase of growth and was strongly enhanced by avicel. The predicted product of cel9 matches the structural architecture of family 9 cellulases such as Thermonospora fusca endo/exocellulase E4. Cel9 protein was synthesized in Escherichia coli from a multicopy plasmid and in Bacillus subtilis from the isopropyl thiogalactoside‐inducible Pspac promoter and was purified from the culture medium. Thermal stability, optimum pH and temperature dependence of Cel9 were similar when expressed from either source, and were indistinguishable from related cellulases produced by thermophilic bacteria. Downstream from cel9 was found a partial ORF, designated cel48, the deduced product of which was highly similar to bacterial exocellobiohydrolases and processive endoglucanases belonging to family 48 of the glycosyl hydrolases. The cel9 and cel48 genes appear to be arranged as part of an operon.
Conserved amino acids at the C‐terminus of rat phospholipase D1 are essential for enzymatic activity
Authors: Xie Zhi , Ho Wan‐Ting , Exton John H.
Published: March 2003

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N‐terminal and C‐terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C‐terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N‐terminal and C‐terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C‐terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C‐terminus of this sequence. Interestingly, the inactive C‐terminal mutants could be complemented by cotransfection with a wild‐type C‐terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C‐terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C‐terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.
GDNF – a stranger in the TGF‐β superfamily?
Authors: Saarma Mart
Published: March 2003

Glial cell line‐derived neurotrophic factor (GDNF) family, consisting of GDNF, neurturin, artemin and persephin are distant members of the transforming growth factor‐β (TGF‐β) superfamily. Unlike other members of the TGF‐β superfamily, which signal through the receptor serine‐threonine kinases, GDNF family ligands activate intracellular signalling cascades via the receptor tyrosine kinase Ret. GDNF family ligands first bind to the glycosylphosphatidylinositol (GPI)‐anchored GDNF family receptor α (GFRα) and then the GDNF family ligand–GFRα complex binds to and stimulates autophosphorylation of Ret. Alternatively, a preassociated complex between GFRα and Ret could form the binding site for the GDNF family ligand. GFRα1, GFRα2, GFRα3 and GFRα4 are the physiological coreceptors for GDNF, neurturin, artemin and persephin, respectively. Although all GDNF family ligands signal via activated Ret, GDNF can signal also via GFRα1 in the absence of Ret. GPI‐anchored GFRα receptors are localized in plasma membrane to lipid rafts. GDNF binding to GFRα1 also recruits Ret to the lipid rafts and triggers association with Src, which is required for effective downstream signalling, leading to differentiation and neuronal survival. GDNF family ligands are potent survival factors for midbrain dopamine neurons, motoneurons, noradrenergic neurons, as well as for sympathetic, parasympathetic and sensory neurons. However, for most neuronal populations, except for motoneurons, TGF‐β is required as a cofactor for GDNF family ligand signalling. Because GDNF and neurturin can rescue dopamine neurons in the animal models of Parkinson disease, as well as motoneurons in vivo, hopes have been raised that GDNF family ligands may be new drugs for the treatment of neurodegenerative diseases. GDNF also has distinct functions outside the nervous system, promoting ureteric branching in kidney development and regulating spermatogenesis.
Mercuric chloride activates the Src‐family protein tyrosine kinase, Hck in myelomonocytic cells
Authors: Robbins Stephen M. , Quintrell Nancy A. , Bishop J. Michael
Published: March 2003

Hck is a member of the Src‐family of protein tyrosine kinases that appears to function in mature leukocytes to communicate a number of extracellular signals including various cytokines. In this study we show that the thiol‐reactive heavy metal, mercuric chloride (HgCl2) induces rapid and robust activation of tyrosine phosphorylation within human myelomonocytic cells. This increase in tyrosine‐phosphorylated proteins requires the activity of Hck because both kinase inactive alleles of Hck and pharmacological inhibitors selective for the Src‐family kinases are able to abrogate the cellular response to HgCl2. Furthermore, ectopic expression of Hck in murine fibroblasts is able to confer HgCl2 responsiveness, as indicated by an increase in tyrosine‐phosphorylated proteins to a normally nonresponsive cell line. Concomitant with the activation of Hck, there is a physical association of Hck with another cytoplasmic protein tyrosine kinase, Syk. The ability of HgCl2 to activate Src‐family kinases such as Hck in hematopoietic cells may help explain why exposure to the heavy metal is associated with immune system dysfunction in rodents as well as humans.
Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D‐amino acid aminotransferase
Authors: Gutierrez Aldo , Yoshimura Tohru , Fuchikami Yoshihiro , Esaki Nobuyoshi
Published: March 2003

The activity and substrate specificity of d‐amino acid aminotransferase (
d‐AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119‐R120‐P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild‐type enzyme in the overall reactions between various amino acids and pyruvate. The presteady‐state kinetic analyses of half‐reactions revealed that the 5‐glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild‐type enzyme towards various amino acids except
d‐aspartate. The 5‐glycine mutant was much more efficient as a catalyst than the wild‐type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except
d‐aspartate and
d‐glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.
Additional PKA phosphorylation sites in human cardiac troponin I
Authors: Ward Douglas G. , Ashton Peter R. , Trayer Hylary R. , Trayer Ian P.
Published: March 2003

We used mass spectrometry to monitor cAMP‐dependent protein kinase catalysed phosphorylation of human cardiac troponin I in vitro. Phosphorylation of isolated troponin I by cAMP‐dependent protein kinase resulted in the covalent incorporation of phosphate on at least five different sites on troponin I, and a S22/23A troponin I mutant incorporated phosphates on at least three sites. In addition to the established phosphorylation sites (S22 and S23) we found that S38 and S165 were the other two main sites of phosphorylation. These ‘overphosphorylation’ sites were not phosphorylated sufficiently slower than S22 and S23 that we could isolate pure S22/23 bisphosphorylated troponin I. Overphosphorylation of troponin I reduced its affinity for troponin C, as measured by isothermal titration microcalorimetry. Phosphorylation of S22/23A also decreased its affinity for troponin C indicating that phosphorylation of S38 and/or S165 impedes binding of troponin I to troponin C. Formation of a troponin I/troponin C complex prior to cAMP‐dependent protein kinase treatment did not prevent overphosphorylation. When whole troponin was phosphorylated by cAMP‐dependent protein kinase, however, [32P]phosphate was incorporated only into troponin I and only at S22 and S23. Mass spectrometry confirmed that overphosphorylation is abolished in the ternary complex. Troponin I bisphosphorylated exclusively at S22 and S23 troponin I showed reduced affinity for troponin C but the effect was diminished with respect to overphosphorylated troponin I. These results show that care should be exercised when interpreting data obtained with troponin I phosphorylated in vitro.
Determination of binding constant of transcription factor AP‐1 and DNA
Authors: Kwon Haeyoung , Park Seyeon , Lee Sangkyou , Lee Dug‐Keun , Yang Chul‐Hak
Published: March 2003

The equilibrium binding and association kinetics of the fos–jun dimer (basic and leucine zipper domain) to the AP‐1 DNA were studied using a quantitative assay. The basic‐region and leucine zipper (bZip) domain of c‐fos was expressed as a fusion protein with glutathione S‐transferase, and it was bound to glutathione‐agarose. The GST‐fused fos bZip region was allowed to form a heterodimer with the bZip domain of c‐jun, to which radiolabeled AP‐1 nucleotides were added. After thorough washing, the gel‐bound radioactivity was counted. The binding and dissociation rate constants (k1 and k‐1) of the fos–jun dimer and DNA could be obtained from a time‐course experiment. The association binding constant (K1) was determined using both a thermodynamic equation and kinetic parameters. Nordihydroguaiaretic acid (NDGA), momordin I, natural product inhibitors of the fos–jun/DNA complex formation, was applied to this jun–GST‐fused fos system and it was found to decrease the apparent equilibrium binding of dimer and DNA. The thermodynamic constant of dimer and inhibitor binding was also determined.
Differential regulation of cAMP‐mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors
Authors: Lee Eun Jin , Lee Soo‐Hyun , Jung Jin‐Won , Lee Weontae , Kim Byung Jin , Park Kye Won , Lim Sung‐Kil , Yoon Chang‐Ju , Baik Ja‐Hyun
Published: March 2003

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G‐protein‐coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP‐responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP‐regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of α‐melanocyte‐stimulating hormone (α‐MSH) at different levels. In our assay of CRE‐mediated gene transcription activity, α‐MSH‐ND was the most efficient α‐MSH analog for MC4R whereas NDP‐MSH was the most efficient for MC3R. Changing the His6 residue of α‐MSH‐ND to Gln or Lys markedly decreased CRE‐mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor–ligand complex by NMR, [Gln6]α‐MSH‐ND and [Lys6]α‐MSH‐ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30–50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor–ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.
Prochiral selectivity in H2O2‐promoted oxidation of arylalkanols catalysed by chloroperoxidase
Authors: Baciocchi Enrico , Fabbrini Maura , Lanzalunga Osvaldo , Manduchi Laura , Pochetti Giorgio
Published: March 2003

The H2O2‐promoted oxidations of (R)‐[α‐2H1]‐and (S)‐[α‐2H1]‐arylalkanols catalysed by chloroperoxidase (CPO) from Caldariomyces fumago have been investigated. It has been found that with (R)‐[α‐2H1]‐alcohols, the oxidation involves almost exclusively the cleavage of the C–H bond, whereas in the case of the oxidation of (S)‐[α‐2H1]‐alcohols, the C–D bond is preferentially broken. These results clearly indicate that the reactions of corresponding undeuterated arylalkanols are characterized by a high prochiral selectivity, involving the cleavage of the pro‐S C–H bond. This prochiral selectivity is poorly influenced by the electronic effect of ring substituents, whereas it decreases with the length of the carbon lateral chain, in the order: benzyl alcohol > 2‐phenylethanol > 3‐phenylpropanol. Molecular binding studies showed that the main factor directing the docking of the substrate in such a specific orientation in the enzyme active site is the interaction between the alcoholic OH group and the residue Glu183. This interaction is likely to drive both the stereochemistry and the regiochemistry of these reactions. A bifurcated hydrogen bond involving the OH group, the carboxylate oxygen of Glu183 and the oxoferryl oxygen might also be operating.