bluuebunny/crossref_metadata_2025 huggingface dataset:
Site‐specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme
Authors: Nishikawa Fumiko , Shirai Miho , Nishikawa Satoshi
Published: March 2003

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single‐stranded regions (SSrA, ‐B and ‐C). The 3D structure of 3′‐cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2′OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site‐specific atomic‐level modifications (such as deoxynucleotides, deoxyribosyl‐2‐aminopurine, deoxyribosylpurine, 7‐deaza‐ribonucleotide and inosine) were incorporated in the smallest trans‐acting HDV ribozyme (47‐mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W‐C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2′‐OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2′‐OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2′‐OH in both the trans‐ and cis‐acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5‐ to 15‐fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.
Dynamic mechanism of nick recognition by DNA ligase
Authors: Cherepanov Alexei V. , de Vries Simon
Published: March 2003

DNA ligases are the enzymes responsible for the repair of single‐stranded and double‐stranded nicks in dsDNA. DNA ligases are structurally similar, possibly sharing a common molecular mechanism of nick recognition and ligation catalysis. This mechanism remains unclear, in part because the structure of ligase in complex with dsDNA has yet to be solved. DNA ligases share common structural elements with DNA polymerases, which have been cocrystallized with dsDNA. Based on the observed DNA polymerase–dsDNA interactions, we propose a mechanism for recognition of a single‐stranded nick by DNA ligase. According to this mechanism, ligase induces a B‐to‐A DNA helix transition of the enzyme‐bound dsDNA motif, which results in DNA contraction, bending and unwinding. For non‐nicked dsDNA, this transition is reversible, leading to dissociation of the enzyme. For a nicked dsDNA substrate, the contraction of the enzyme‐bound DNA motif (a) triggers an opened–closed conformational change of the enzyme, and (b) forces the motif to accommodate the strained A/B‐form hybrid conformation, in which the nicked strand tends to retain a B‐type helix, while the non‐nicked strand tends to form a shortened A‐type helix. We propose that this conformation is the catalytically competent transition state, which leads to the formation of the DNA–AMP intermediate and to the subsequent sealing of the nick.
Rodent α‐chymases are elastase‐like proteases
Authors: Kunori Yuichi , Koizumi Masahiro , Masegi Tsukio , Kasai Hidenori , Kawabata Hiroshi , Yamazaki Yuzo , Fukamizu Akiyoshi
Published: March 2003

Although the α‐chymases of primates and dogs are known as chymotrypsin‐like proteases, the enzymatic properties of rodent α‐chymases (rat mast cell protease 5/rMCP‐5 and mouse mast cell protease 5/mMCP‐5) have not been fully understood. We report that recombinant rMCP‐5 and mMCP‐5 are elastase‐like proteases, not chymotrypsin‐like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease‐5s (MCP‐5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site‐directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP‐5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase‐like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP‐5. Structural models of mMCP‐5 and the V216G mutant based on the crystal structures of serine proteases (rMCP‐2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent α‐chymases have unique biological activity different from the chymases of other species.
Role of tyrosine 238 in the active site of Rhodotorula gracilisd‐amino acid oxidase
Authors: Boselli Angelo , Sacchi Silvia , Job Viviana , Pilone Mirella S. , Pollegioni Loredano
Published: March 2003

Y238, one of the very few conserved residues in the active site of d‐amino acid oxidases (DAAO), was mutated to phenylalanine and serine in the enzyme from the yeast Rhodotorula gracilis. The mutated proteins are catalytically competent thus eliminating Tyr238 as an active‐site acid/base catalyst. Y238F and Y238S mutants exhibit a threefold slower turnover on d‐alanine as substrate, which can be attributed to a slower rate of product release relative to the wild‐type enzyme (a change of the rate constants for substrate binding was also evident). The Y238 DAAO mutants have spectral properties similar to those of the wild‐type enzyme but the degree of stabilization of the flavin semiquinone and the redox properties in the free form of Y238S are different. The binding of the carboxylic acid competitive inhibitors and the substrate d‐alanine are changed only slightly, suggesting that the overall substrate binding pocket remains intact. In agreement with data from the pH dependence of ligand binding and with the protein crystal structure, site‐directed mutagenesis results emphasize the importance of residue Y238 in controlling access to the active site instead of a role in the substrate/ligand interaction.
Signaling events mediating activation of brain ethanolamine plasmalogen hydrolysis by ceramide
Authors: Latorre Eduardo , Collado M. Pilar , Fernández Inmaculada , Aragonés M. Dolores , Catalán R. Edgardo
Published: March 2003

Ceramide is a lipid second messenger that acts on multiple‐target enzymes, some of which are involved in other signal‐transduction systems. We have previously demonstrated that endogenous ceramide modifies the metabolism of brain ethanolamine plasmalogens. The mechanism involved was studied. On the basis of measurements of breakdown products, specific inhibitor effects, and previous findings, we suggest that a plasmalogen‐selective phospholipase A2 is the ceramide target. Arachidonate‐rich pools of the diacylphosphatidylethanolamine subclass were also affected by ceramide, but the most affected were plasmalogens. Concomitantly with production of free arachidonate, increased 1‐O‐arachidonoyl ceramide formation was observed. Quinacrine (phospholipase A2 inhibitor) and 1‐O‐octadecyl‐2‐O‐methyl‐rac‐glycerol‐3‐phosphocholine (CoA‐independent transacylase inhibitor) prevented all of these ceramide‐elicited effects. Therefore, phospholipase and transacylase activities are tightly coupled. Okadaic acid (phosphatase 2A inhibitor) and PD 98059 (mitogen‐activated protein kinase inhibitor) modified basal levels of ceramide and sphingomyelinase‐induced accumulation of ceramide, respectively. Therefore, they provided no evidence to determine whether there is a sensitive enzyme downstream of ceramide. The evidence shows that there are serine‐dependent and thiol‐dependent enzymes downstream of ceramide generation. Furthermore, experiments with Ac‐DEVD‐CMK (caspase‐3 specific inhibitor) have led us to conclude that caspase‐3 is downstream of ceramide in activating the brain plasmalogen‐selective phospholipase A2.
tmRNA from Thermus thermophilus
Authors: Stepanov Victor G , Nyborg Jens
Published: March 2003

The interaction of a Thermus thermophilus tmRNA transcript with alanyl‐tRNA synthetase and elongation factor Tu has been studied. The synthetic tmRNA was found to be stable up to 70 °C. The thermal optimum of tmRNA alanylation was determined to be around 50 °C. At 50 °C, tmRNA transcript was aminoacylated by alanyl‐tRNA synthetase with 5.9 times lower efficiency (kcat/Km) than tRNAAla, primarily because of the difference in turnover numbers (kcat). Studies on EF‐Tu protection of Ala∼tmRNA against alkaline hydrolysis revealed the existence of at least two different binding sites for EF‐Tu on charged tmRNA. The possible nature of these binding sites is discussed.
Probing suggested catalytic domains of glycosyltransferases by site‐directed mutagenesis
Authors: Hefner Tobias , Stöckigt Joachim
Published: March 2003

The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1β family of glycosyltransferases. This enzyme was used to prove, by site‐directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme‐catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity, does not serve as the nucleophile in the catalytic centre as proposed. When it is replaced by Ala, the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin. Enzyme activities of wild‐type and E368A towards several substrates were not affected at the same level. His360 at position 1 of NRD1β glycosyltransferases occupies a more crucial role as expected. When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases ≈ 1000‐fold. Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an ≈ 4000‐fold lower activity compared with the wild‐type. This mutein still produces a β‐glucoside, not an α‐glucoside and therefore indicates that generation of the typical E–E motif of NRD1α glycosyltransferases does not convert a NRD1β enzyme into a NRD1α enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.
Two CCAAT/enhancer binding protein sites in the cytochrome P4503A1 locus
Authors: Rodrigues Elsa , Vilarem Marie‐José , Ribeiro Vera , Maurel Patrick , Lechner Maria C.
Published: March 2003

Induction of CYP3A genes by the ligand‐activated pregnane‐X‐receptor (PXR) involves the interaction of other as yet unidentified liver transcription factors. Here we show that the CYP3A1 promoter contains two active sites controlled by the CCAAT/enhancer‐binding protein α (C/EBPα), previously shown to regulate a number of liver stress response genes. We have identified two functional C/EBP binding sites at the CYP3A1 promoter that confer luciferase activity to C/EBPα cotransfected CHO cells. When inserted upstream of a thymidine kinase promoter, oligonucleotides corresponding to these elements (−350/−311 and −628/−608), increase reporter gene expression when cotransfected with a C/EBPα expression vector. Point mutations in the most conserved nucleotides in either element prevent binding of C/EBPα and abolish transactivation of the CYP3A1 promoter. Moreover, we demonstrate that C/EBPα accumulates in the rat liver nuclei in response to dexamethasone, and that under these conditions C/EBPα binds to the CYP3A1 promoter elements. Our results suggest a correlation between transcription of C/EBPα, nuclear protein function and induction of CYP3A1 by dexamethasone in the liver. They also support the notion that C/EBPα participates in the up‐regulation of the CYP3A1 gene in response to synthetic glucocorticoids.
Functionally distinct dopamine and octopamine transporters in the CNS of the cabbage looper moth*
Authors: Gallant Pamela , Malutan Tabita , McLean Heather , Verellen LouAnn , Caveney Stanley , Donly Cam
Published: March 2003

A cDNA was cloned from the cabbage looper Trichoplusia ni based on similarity to other cloned dopamine transporters (DATs). The total nucleotide sequence is 3.8 kb in length and contains an open reading frame for a protein of 612 amino acids. The predicted moth DAT protein (TrnDAT) has greatest amino acid sequence identity with Drosophila melanogasterDAT (73%) and Caenorhabditis elegansDAT (51%). TrnDAT shares only 45% amino acid sequence identity with an octopamine transporter (TrnOAT) cloned recently from this moth. The functional properties of TrnDAT and TrnOAT were compared through transient heterologous expression in Sf9 cells. Both transporters have similar transport affinities for DA (Km 2.43 and 2.16 µm, respectively). However, the competitive substrates octopamine and tyramine are more potent blockers of [3H]dopamine (DA) uptake by TrnOAT than by TrnDAT. D‐Amphetamine is a strong inhibitor and l‐norepinephrine a weak inhibitor of both transporters. TrnDAT‐mediated DA uptake is approximately 100‐fold more sensitive to selective blockers of vertebrate transporters of dopamine and norepinephrine, such as nisoxetine, nomifensine and dibenzazepine antidepressants, than TrnOAT‐mediated DA uptake. TrnOAT is 10‐fold less sensitive to cocaine than TrnDAT. None of the 15 monoamine uptake blockers tested was TrnOAT‐selective. In situ hybridization shows that TrnDAT and TrnOAT transcripts are expressed by different sets of neurons in caterpillar brain and ventral nerve cord. These results show that the caterpillar CNS contains both a phenolamine transporter and a catecholamine transporter whereas in the three invertebrates whose genomes have been completely sequenced only a dopamine‐selective transporter is found.
Purification and characterization of three isoforms of chrysophsin, a novel antimicrobial peptide in the gills of the red sea bream, Chrysophrys major
Authors: Iijima Noriaki , Tanimoto Norio , Emoto Yohko , Morita Yohko , Uematsu Kazumasa , Murakami Tomoya , Nakai Toshihiro
Published: March 2003

We report here the isolation of three isoforms of a novel C‐terminally amidated peptide from the gills of red sea bream, Chrysophrys (Pagrus) major. Peptide sequences were determined by a combination of Edman degradation, MS and HPLC analysis of native and synthetic peptides. Three peptides, named chrysophsin‐1, chrysophsin‐2, and chrysophsin‐3, consist of 25, 25, and 20 amino acids, respectively, and are highly cationic, containing an unusual C‐terminal RRRH sequence. The α‐helical structures of the three chrysophsin peptides were predicted from their secondary structures and were confirmed by CD spectroscopy. The synthetic peptides displayed broad‐spectrum bactericidal activity against Gram‐negative and Gram‐positive bacteria including Escherichia coli, Bacillus subtilis, and fish and crustacean pathogens. The three peptides were also hemolytic. Immunohistochemical analysis showed that chrysophsins were localized in certain epithelial cells lining the surface of secondary lamellae and eosinophilic granule cell‐like cells at the base of the secondary lamellae in red sea bream gills. Their broad ranging bactericidal activities, combined with their localization in certain cells and eosinophilic granule cell‐like cells in the gills, suggest that chrysophsins play a significant role in the innate defense system of red sea bream gills.
Complex alternative splicing of the hKLK3 gene coding for the tumor marker PSA (prostate‐specific‐antigen)
Authors: Heuzé‐Vourc'h Nathalie , Leblond Valérie , Courty Yves
Published: March 2003

PSA (prostate‐specific antigen), the most useful serum marker for prostate cancer, is encoded by the hKLK3 gene and is present in the serum as a mixture of several molecular species. This work was performed to identify the hKLK3 transcripts in order to determine how many proteins resembling PSA are synthesized from the hKLK3 gene and secreted in blood. Combined Northern blotting, molecular cloning and database searching showed that the hKLK3 gene produces at least 15 transcripts ranging in size from 0.7 to 6.1 kb. Polysomal distribution analysis revealed that the transcripts shorter than 3.1 kb are efficiently translated in prostate cell line. A total of 12 hKLK3 transcripts have been completely or partially cloned. They result from alternative splicing or/and alternative polyadenylation involving complex regulation. They code for eight proteins: PSA, a truncated form of PSA (PSA‐Tr), five PSA variants (PSA‐RPs) and one protein (PSA‐LM) unrelated to PSA. Using a specific antibody, we detected the PSA‐RP2 variant in prostate tissue. All the variants share the same signal peptide and could contribute to the diversity of hKLK3 proteins in prostate fluid and blood.
Characterization of acetyl‐CoA/propionyl‐CoA carboxylase in Metallosphaera sedula
Authors: Hügler Michael , Krieger Robert S. , Jahn Martina , Fuchs Georg
Published: March 2003

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3‐hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP‐dependent carboxylations of acetyl‐CoA and propionyl‐CoA to malonyl‐CoA and methylmalonyl‐CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl‐CoA and propionyl‐CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl‐CoA/propionyl‐CoA carboxylase from M. sedula is active at 75 °C and is isolated as a stabile functional protein complex of 560 ± 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (α) and carboxytransferase (γ), respectively, and a small 18.6 kDa biotin carrier protein (β). These subunits probably form an (αβγ)4 holoenzyme. It has a catalytic number of 28 s−1 at 65 °C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl‐CoA, 0.07 mm for propionyl‐CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl‐CoA/propionyl‐CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.
Biochemical characterization of a U6 small nuclear RNA‐specific terminal uridylyltransferase
Authors: Trippe Ralf , Richly Holger , Benecke Bernd‐Joachim
Published: March 2003

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3′‐end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3′‐terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3′‐terminal UMP residues were filled up efficiently, generating the 3′‐terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill‐in reaction by the U6‐TUTase was not controlled by opposite‐strand nucleotides, excluding an RNA‐dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility‐shift analyses showed that the U6‐TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6‐TUTase.
The catalytic role of the distal site asparagine‐histidine couple in catalase‐peroxidases
Authors: Jakopitsch Christa , Auer Markus , Regelsberger Günther , Jantschko Walter , Furtmüller Paul G. , Rüker Florian , Obinger Christian
Published: March 2003

Catalase‐peroxidases (KatGs) are unique in exhibiting an overwhelming catalase activity and a peroxidase activity of broad specificity. Similar to other peroxidases the distal histidine in KatGs forms a hydrogen bond with an adjacent conserved asparagine. To investigate the catalytic role(s) of this potential hydrogen bond in the bifunctional activity of KatGs, Asn153 in Synechocystis KatG was replaced with either Ala (Asn153→Ala) or Asp (Asn153→Asp). Both variants exhibit an overall peroxidase activity similar with wild‐type KatG. Cyanide binding is monophasic, however, the second‐order binding rates are reduced to 5.4% (Asn153→Ala) and 9.5% (Asn153→Asp) of the value of native KatG [(4.8 ± 0.4) × 105 m−1·s−1 at pH 7 and 15 °C]. The turnover number of catalase activity of Asn153→Ala is 6% and that of Asn153→Asp is 16.5% of wild‐type activity. Stopped‐flow analysis of the reaction of the ferric forms with H2O2 suggest that exchange of Asn did not shift significantly the ratio of rates of H2O2‐mediated compound I formation and reduction. Both rates seem to be reduced most probably because (a) the lower basicity of His123 hampers its function as acid‐base catalyst and (b) Asn153 is part of an extended KatG‐typical H‐bond network, the integrity of which seems to be essential to provide optimal conditions for binding and oxidation of the second H2O2 molecule necessary in the catalase reaction.
Activation of STAT6 is not dependent on phosphotyrosine‐mediated docking to the interleukin‐4 receptor and can be blocked by dominant‐negative mutants of both receptor subunits
Authors: Moriggl Richard , Erhardt Ingrid , Kammer Winfried , Berchtold Susanne , Schnarr Bernd , Lischke Antje , Groner Bernd , Friedrich Karlheinz
Published: March 2003

Stimulation of susceptible cells by interleukin‐4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence‐specific transcription factor. We functionally reconstituted human interleukin‐4 receptor complexes with intracellular truncations of either the α or γ subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin‐4. By assaying the signalling properties of human interleukin‐4‐receptor α‐chain‐deletion constructs in both Ba/F3 cells and COS‐7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin‐4 receptor was completely abolished by disruption of the membrane‐proximal ’box 1' motif in the interleukin‐4 receptor α chain. Our results indicate a redundant role of the previously defined phosphotyrosine‐containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor‐associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin‐4 receptor subunits are able to block interleukin‐4 induced signalling via STAT6.
Characterization of a methyljasmonate‐inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves
Authors: Vörös Kirsten , Feussner Ivo , Kühn Hartmut , Lee Justin , Graner A. , Löbler Marian , Parthier Benno , Wasternack Claus
Published: March 2003

We found three methyl jasmonate−induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX‐92, ‐98 and ‐100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7, 949−957]. At least two of them (LOX‐92 and LOX‐100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX‐100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2‐type lipoxygenase and was therefore designated LOX2 : Hv : 1. The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13‐LOX and arachidonate 15‐LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2 : Hv : 1, suggesting a separate signalling pathway compared with other jasmonate‐induced proteins of barley. The properties of LOX2 : Hv : 1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.
Role of carbohydrate and protein in the binding of tissue‐type plasminogen activator to the human mannose receptor
Authors: Noorman Femke , Barrett‐Bergshoeff Marrie M. , Rijken Dingeman C.
Published: March 2003

The 175‐kDa mannose receptor is one of the receptors that mediates the clearance of tissue‐type plasminogen activator (t‐PA). The affinity of t‐PA for the mannose receptor is much higher than the affinity of other high‐mannose‐type oligosaccharide‐containing glycoproteins. In order to find an explanation for this high affinity, we studied the biochemical interaction of various forms of t‐PA with the isolated human mannose receptor in several in vitro binding assays.

t‐PA showed a high affinity (Ki = 0.2 nM) for the mannose receptor and the interaction could be fully inhibited by mannan or polyclonal antibodies against the mannose receptor. The interaction was not affected by non‐glycosylated t‐PA. The high affinity differed slightly between t‐PAs synthesized by various cell types (range Ki 0.2−0.7 nM) and between various glycoforms of t‐PA. No statistically significant difference in affinity between t‐PA and t‐PA complexed to inhibitors was observed. In contrast to intact t‐PA, a trypsin digest of t‐PA had a low affinity (Ki = 0.5 μM) for the mannose receptor. Both intact and trypsin digests of the high‐mannose‐type oligosaccharide‐containing glycoproteins ribonuclease B and ovalbumin had a low affinity (Ki 0.5−1.5 μM) for the mannose receptor.

We conclude that neither protein−protein interactions, nor the complex‐type oligosaccharides and the fucose residue on t‐PA contribute significantly to the high‐affinity binding of t‐PA. We suggest that the conformation of the high‐mannose‐type oligosaccharide on t‐PA is influenced by the protein moiety of t‐PA in such a way that the oligosaccharide has a high affinity for the mannose receptor.
Split‐receptors in the tachykinin neurokinin‐1 system
Authors: Nielsen Soren Møller , Elling Christian E. , Schwartz Thue W.
Published: March 2003

Several membrane proteins have been functionally expressed from non‐covalently coupled, contiguous segments especially with the split‐site located between natural domains. Experiments using such ’split‐proteins' were here performed in the tachykinin neurokinin‐1 (NK1) receptor with co‐expression of contiguous segments with split‐sites positioned in various intracellular and extracellular loops. The construct where the split‐site was located in intracellular loop 3 gave a reasonable expression level of substance‐P‐binding sites, i.e. 12 % of wild‐type expression. Of the other split‐receptors tested, only the one with the split‐site located just outside transmembrane (TM) segment‐V gave any detectable substance P binding, which however only was 1 % of the wild‐type expression level. The construct with the split‐site located in intracellular loop 3 bound all of the tested peptide agonists and non‐peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non‐split NK1 receptor, the affinity for neither substance P nor for the prototype non‐peptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split‐receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK, receptor is preferentially reconstituted by co‐expression of a putative A‐domain including TM‐I−V and a B‐domain including TM‐VI and ‐VII. It is suggested that a number of rhodopsin‐like 7TM receptors may function as two‐domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.
Cytochrome‐c−binding site on cytochrome oxidase in Paracoccus denitrificans
Authors: Witt Heike , Malatesta Francesco , Nicoletti Flavia , Brunori Maurizio , Ludwig Bernd
Published: March 2003

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site‐directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady‐state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped‐flow measurements. Increasing the ionic strength, the maximum of the bell‐shaped dependence of the steady‐state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady‐state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre‐steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.
Dephosphorylation activates the purified plant plasma membrane H+‐ATPase
Authors: Desbrosses Guilhem , Stelling Joërg , Renaudin Jean‐Pierre
Published: March 2003

The plasma membrane H+‐ATPase was purified from tobacco cells (line BY‐2). After solubilization by lysophosphatidylcholine followed by separation on a glycerol gradient, a fraction with a high specific activity of 9 μmol ATP min−1 mg protein−1 was obtained, in which the H+‐ATPase polypeptide represented at least 80 % of the protein. The incubation of this fraction in the presence of alkaline phosphatase increased H+‐ATPase activity by 40 %, in a manner consistent with dephosphorylation of the enzyme itself. The hydrolytic activity of the solubilized enzyme and its proton translocating activity, after reconstitution into proteoliposomes, were stimulated to the same extent. Alkaline phosphatase treatment was also accompanied by a 92 % decrease in the H+‐ATPase phosphothreonine content, whereas the phosphoserine residues were almost unaffected. The dephosphorylation induced a slight decrease of the affinity of the enzyme towards ATP. The purified enzyme was not activated by lysophosphatidylcholine addition nor by trypsin‐mediated proteolysis, two treatments reported to release the inhibitory control by the C‐terminal domain of the H+‐ATPase and to increase the affinity of the enzyme towards ATP. Based on these results, the regulatory phosphorylation evoked by alkaline phosphatase most likely differs from the autoinhibitory control of the H+‐ATPase by its C‐terminal domain.
Divalent cation dependence and inhibition of Schistosoma mansoni ATP diphosphohydrolase by fluorosulfonylbenzoyl adenosine
Authors: Torres Christiane R. , Vasconcelos Eveline G. , Ferreira Sérgio T. , Verjovski‐Almeida Sergio
Published: March 2003

Hydrolysis of ATP or ADP catalyzed by the ATP diphosphohydrolase of Schistosoma mansoni tegument was measured in the presence of different cations. ATP diphosphohydrolase was stimulated by micromolar concentrations of either Ca2+ or Mg2+, Ca2+ producing threefold higher maximal activities than Mg2+. Kinetic studies indicated that Ca2+ and Mg2+ compete for the same binding site on the enzyme. The effect of covalent labeling of ATP diphosphohydrolase with the ATP analog fluorosulfonylbenzoyl adenosine (FSO2BzAdo) was studied. Schistosome tegument was passed through with Sephadex G‐50 filtration centrifugation columns to remove endogenous nucleotides, and this was followed by labeling with FSO2BzAdo. Incubation of ATP diphosphohydrolase with 1 mM FSO2BzAdo for 1 h inhibited ATPase or ADPase activities by 60 % and 50 %, respectively. Addition of ATP together with FSO2BzAdo provided greater than 90 % protection against FSO2BzAdo inactivation, indicating that FSO2BzAdo binds to an ATP‐binding site on the ATP diphosphohydrolase. Furthermore, addition of FSO2BzAdo to a medium containing intact worms caused 30 % and 50 % inhibition of ATPase and ADPase activities, respectively, indicating that the ATP‐binding site of diphosphohydrolase is accessible to FSO2BzAdo from the external surface of S. mansoni worms.
Phenylarsine oxide as an inhibitor of the activation of the neutrophil NADPH oxidase
Authors: Doussiere Jacques , Poinas Alexandra , Blais Chrystèle , Vignais Pierre V.
Published: March 2003

Plasma membranes of neutrophil cells contain the redox component of the O.es−.rb.ei2.rb‐generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an α subunit of 22 kDa and a β subunit of 85−105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane‐bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell‐free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell‐free system. To determine the PAO‐binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4‐[N‐(4‐azido‐2‐nitrophenyl)amino‐[3H]acetamido]phenylarsine oxide, ([3H]azido‐PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido‐PAO resulted in the prominent labeling of a protein of 85−105 kDa whose migration on SDS/PAGE coincided with that of the β subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85−105 kDa was shifted to 50−60 kDa as was the immunodetected β subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the β subunit of the membrane‐bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell‐free system. Incorporation of [3H]azido‐PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the β subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.
Cluster‐iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states
Authors: Iametti Stefania , Uhlmann Heike , Ragg Enzio , Sala Nicla , Grinberg Asya , Beckert Vita , Bernhardt Rita , Bonomi Francesco
Published: March 2003

Site‐directed mutants of adrenodoxin were studied for their ability to undergo cluster‐iron substitution when reacted with zinc or cadmium salts under non‐denaturing conditions in the presence or absence of reductants. Equilibrium and kinetic data for metal substitution were correlated with data on the stability to thermal unfolding and with the redox potential of the protein. Similarly to the wild‐type protein, all mutants were able to stabilize a substituted form of the protein containing two metal (Zn or Cd) atoms and two sulfide ions/mol protein and a substituted form of the protein containing two sulfide ions and five Cd atoms/mol protein. However, the distribution of these two metal‐substituted forms was different among the investigated proteins. [Ser95]Adrenodoxin stabilized either metal‐substituted forms, confirming that Cys95 is not involved in metal coordination, even when five Cd atoms are bound to the protein. Removal of the extremely conserved hydroxy function at position 54 resulted in complete apoprotein formation upon reaction with Cd (75 % with Zn) under reducing conditions, indicating a cluster‐harboring role for this function, which is conserved in all known 2Fe‐2S proteins. Mutants at His56, which represents a residue unique to most vertebrate‐type ferredoxins, were much more reactive than the wild‐type protein with either metal, indicating that His56 plays a prominent role in the stabilization of the protein structure in the immediate vicinity of the cluster in this class of proteins. The nature of the metal‐substitution products was dependent on cluster accessibility. For the reduced proteins, apoprotein formation depended on protein stability, while the velocity of metal substitution depended on the ease of cluster reduction.
Autonomous folding of the recombinant large cytoplasmic loop of sarcoplasmic reticulum Ca2+‐ATPase probed by affinity labeling and trypsin digestion
Authors: Moutin Marie‐Jo , Rapin Catherine , Miras Roger , Vinçon Mathilde , Dupont Yves , McIntosh David B.
Published: March 2003

Recombinant large cytoplasmic loop (LCL, residues 329−740) of sarcoplasmic reticulum Ca2+‐ATPase, expressed in and purified from Escherichia coli, comprises most of the active site and binds ATP [Moutin, M.‐J., Cuillel, M., Rapin, C., Miras, R., Anger, M., Lompré, A.‐M. & Dupont, Y. (1994) J. Biol. Chem. 269, 11 147−11 154]. In this study, we show that fluorescein‐5′ isothiocyanate (FITC) specifically labels the same lysine residue as in the native Ca2+‐ATPase (Lys515), with similar kinetics and pH dependence. ATP blocks the reaction with the lysine residue, but at higher concentrations compared with those for the native pump, in agreement with the lower ATP‐binding affinity found previously. Graded tryptic digestion of LCL shows that favored cleavage is at the T1 site and that the N‐terminal 75 % of LCL are resistant to trypsin, as is native Ca2+‐ATPase.

Other experiments reveal differences to the native pump. (a) FITC derivatizes some ‐SH groups of LCL. (b) The C‐terminal 25 % of the polypeptide is susceptible to end‐clipping by trypsin. (c) 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP fails to specifically label the LCL (on the equivalent of Lys492), although it binds tightly (KD = 1.3 μM) and (d) Glutaraldehyde does not specifically cross‐link LCL (between the equivalent of Lys492 and Arg678). These results could be explained by a flexible and loose structure of the hinge region of LCL (C‐terminal 25 %). Anchoring this region in the membrane and/or interaction with the missing β‐strand domain may be required for its compact folding and proper interaction with the rest of LCL.

The results suggest that the N‐terminal 75 % of LCL expressed in E. coli folds autonomously to a fairly stable unit and native‐like structure, encompassing the phosphorylation and central ATP binding sections. The hinge region does not appear to be part of the FITC‐binding site but constitutes portions of the 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐ATP and, probably, ATP‐binding site.
The penicillin regulator PENR1 of Aspergillus nidulans is a HAP‐like transcriptional complex
Authors: Litzka Olivier , Papagiannopolous Peter , Davis Meryl A. , Hynes Michael J. , Brakhage Axel A.
Published: March 2003

In Aspergillus nidulans, a DNA‐binding complex, PENR1, was shown to bind to two CCAAT‐box‐containing DNA elements located in the promoter regions of the bidirectionally oriented penicillin biosynthesis genes acvA and ipnA, and of the aat promoter. Here, partial purification of PENR1 and western blotting using anti‐HAPC sera indicated that the previously identified HAPC protein, which was suggested to be part of the CCAAT‐binding complex AnCF, is also part of PENR1. This was confirmed by band shift assays using protein extracts of a ΔhapC strain which exhibited no PENR1 DNA‐binding activity. Supershift assays and immunoprecipitation analysis using anti‐HAPC sera provided evidence that HAPC is part of the PENR1 complex. In ΔhapC strains, penicillin production was reduced, as was expression of both an ipnA‐lacZ and aat‐lacZ gene fusion. Hence, HAPC‐containing PENR1 appears to act as an activator on ipnA and aat expression. However, deletion of hapC had little effect on acvA expression during a fermentation run in fermentation medium. Previous results which had shown that specific deletion of the PENR1‐binding site between acvA and ipnA resulted in a strong increase of expression of an acvA‐uidA gene fusion, together with the present data, suggest the possibility of the existence of a repressor protein that binds close to or overlaps the PENR1‐binding site. It is also shown that binding of PENR1 induced bending of a DNA fragment spanning the PENR1‐binding site between acvA and ipnA in vitro.
The biosynthesis of 8‐O‐methylated sialic acids in the starfish Asterias rubens
Authors: Kelm Antje , Shaw Lee , Schauer Roland , Reuter Gerd
Published: March 2003

The unusual, 8‐O‐methylated sialic acids have only been found in glycoconjugates of certain species of starfish. Using a detergent‐solubilised extract of a particulate fraction from gonads of Asterias rubens, a specific methylation of endogenous and exogenous glycoconjugate‐bound sialic acids was detected with [14C]‐S‐adenosyl‐
L‐methionine ([14C]AdoMet) as the methyl donor. For this test, a filtration assay was developed using glutardialdehyde‐fixed horse erythrocyte membranes as methyl acceptor. The enzyme catalysing this reaction, the sialate‐8‐O‐methyltransferase, was purified 22 000‐fold in a yield of 13 % by ion‐exchange chromatography and two cycles of affinity chromatography on S‐adenosyl‐
L‐homocysteine−Sepharose. Upon SDS/PAGE under reducing conditions, the purified sialate‐8‐O‐methyltransferase revealed two bands with apparent molecular masses of 58 kDa and 62 kDa. Since no evidence for the presence of subunits was found, the relationship between these two species is unknown. The enzyme was optimally active over a broad range of pH (7.4−8.3) and at 37 +C. After EDTA treatment, restoration of the activity depended, in contrast to most methyltransferases, on Mn2+ or Co2+, the latter to a lesser extent. Although both, free N‐acetylneuraminic acid and N‐glycoloylneuraminic acid were methylated, sialic acids α‐glycosidically bound to a number of oligosaccharides and glycoproteins were better substrates. In the presence of 20 μM AdoMet, apparent Km values of 299 μM and 44 μM were found for N‐acetylneuraminic acid and N‐acetylneuraminyl α2,3‐lactose, respectively. Using N‐acetylneuraminyl α2,3lactose as acceptor, an apparent Km of 7.1 μM was found for S‐adenosyl‐
L‐methionine. Therefore, the sialate‐8‐O‐methyltransferase is the first enzyme known to modify both free and glycoconjugate‐bound sialic acids.
Structural analysis of the O‐antigenic polysaccharide from Vibrio mimicus N‐1990
Authors: Landersjö Clas , Weintraub Andrej , Ansaruzzaman Mohammed , Albert M. John , Widmalm Göran
Published: March 2003

The O‐antigenic polysaccharide part of the lipopolysaccharide from Vibrio mimicus N‐1990 has been investigated. Sugar and methylation analysis of native and dephosphorylated polysaccharide together with NMR spectroscopy show that the polysaccharide is composed of tetrasaccharide repeating units. The structure of the repeating unit of the polysaccharide from V. mimicus N‐1990 could be determined as :

←4)‐α‐ D‐GalpNAc‐(1←3)‐β‐
D‐GalpNAc‐(1←2)‐4,6‐P‐β‐
D‐Galp‐(1←3)‐α‐
D‐GalpNAc‐(1←  

The V. mimicus N‐1990 strain cross‐reacts with antibodies elicited against the Vibrio cholerae O139. The nature of this cross‐reactivity resides in the partial structure comprising the galactosyl residue substituted with a cyclic phosphate. This element is present in the cell‐wall‐associated polysaccharides of both strains.
Purification and properties of the 5′‐3′ exonuclease D190 ←A mutant of DNA polymerase I from Streptococcus pneumoniae
Authors: Amblar Mónica , López Paloma
Published: March 2003

A D190 ←A mutation was introduced at the 5′‐3′ exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site‐directed mutagenesis of the polA gene. Comparison of the S. pneumoniae DNA polymerase I, its polymerase domain, and the [Ala190]exonuclease mutant revealed that the mutant polypeptide retains the polymerase activity of the parental enzyme and displayed the strand‐displacement activity of its polymerase domain. However, introduction of the mutation resulted in a 2500‐fold reduction of the 5′‐3′ exonuclease catalytic rate compared with the wild‐type enzyme. Moreover, the mutation at the Asp190 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5′‐3′ exonucleolytic activity. These results provide experimental support for a direct involvement of this aspartic acid residue in a metal‐assisted 5′‐3′ exonucleolytic reaction in type‐I‐like bacterial DNA polymerases and related bacteriophage 5′‐3′ exonucleases.
The N‐termini of the α and β subunits at the top of F1 stabilize the energy‐transfer function in the mitochondrial F1Fo ATP synthase
Authors: Xu Ting , Candita Cosimo , Amoruso Giovanna , Papa Sergio
Published: March 2003

Limited trypsin digestion of isolated F1 removed 15 and 7 amino acids from the N‐termini of the α and β subunits respectively and left other subunits untouched as shown by electrophoresis, immunoblotting and protein sequencing. The cooperativity for ATP hydrolysis by soluble F1 was impaired by trypsin digestion. The Km2 obtained from Eadie‐Hofstee plots apparently decreased in trypsin‐digested F1 but the affinity for adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[NH]P) and GTP hydrolysis was not influenced. The inhibition of ATP hydrolysis by ADP was attenuated by trypsin digestion. Trypsin digestion of F1 did not affect its capacity to bind to Fo nor did it alter the sensitivity of ATP hydrolysis in the F1Fo reconstituted system to oligomycin and N,N′‐dicyclohexylcarbodiimide. The cleavage of the α and β subunits did, on the other hand impair : (a) the ATP‐driven proton pumping in the reconstituted F1Fo complex; (b) the inhibition by F1 of passive proton conduction in Fo; (c) the inhibition of passive proton conduction in Fo by AdoPP[NH]P binding to F1. These results show that the limited cleavage of the N‐termini of the α and β subunits, located on the top of F1, results in decoupling of catalysis from proton transport. The possible relationship of these observations with the binding change rotatory model of the F1Fo ATP synthase is discussed.