bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus
Authors: Wang Ruigong , Lee So Young , Cerenius Lage , Söderhäll Kenneth 
Published: March 2003

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the β‐1,3‐glucan triggered activation of prophenoloxidase in vitro. The C‐terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N‐terminal half contains a cationic glycine‐rich domain, a cationic proline‐rich domain and a clip‐domain, in which the disulfide‐bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian β‐defensins. Antibodies made against both the C‐ and the N‐terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti‐C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti‐N to react with the N‐terminal half of proppA. The recombinant clip‐domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram‐positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 µm and 17.9 µm, respectively. These results suggest that the clip‐domains in proppAs may function as antibacterial peptides.
Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage
Authors: Portaro Fernanda C. V. , Hayashi Mirian A. F. , Silva Celio L. , de Camargo Antonio C. M. 
Published: March 2003

The fate of the proteasome‐generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide‐degrading and ‐binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257−264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo‐peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal‐generated oligopeptides in the cytosol.
Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Gonzalez Leticia , Phalip Vincent , Zhang Cheng‐Cai 
Published: March 2003

Eukaryotic‐like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen‐fixing and heterocyst‐forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic‐like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.
The uptake by cells of 2‐arachidonoylglycerol, an endogenous agonist of cannabinoid receptors
Authors: Bisogno Tiziana , Maccarrone Mauro , De Petrocellis Luciano , Jarrahian Abbas , Finazzi‐Agrò Alessandro , Hillard Cecilia , Di Marzo Vincenzo 
Published: March 2003

It is not yet clear if the endocannabinoid 2‐arachidonoylglycerol (2‐AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N‐arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2‐AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2‐AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 ± 2.0 and 15.3 ± 3.1 µm, Bmax 1.70 ± 0.30 and 0.24 ± 0.04 nmol·min−1·mg protein−1, respectively). Despite these similar Km values, 2‐AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 ± 3.9 µm) significantly higher than those required to either 2‐AG or AEA to inhibit [3H]2‐AG uptake (Ki = 18.9 ± 1.8 and 20.5 ± 3.2 µm, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2‐AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2‐AG alone; (b) the uptake of [14C]AEA and [3H]2‐AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 ± 0.7 and 10.2 ± 1.7 µm, respectively) and linvanil (Ki = 9.5 ± 0.7 and 6.4 ± 1.2 µm, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2‐AG, thus suggesting that 2‐AG action can be regulated through NO release; (d) AEA and 2‐AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.
Human placental calreticulin
Authors: Højrup Peter , Roepstorff Peter , Houen Gunnar 
Published: March 2003

The domain organization and the post‐translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6‐ to 7‐kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N‐terminus were observed, but no stable fragments of intermediate size were found. These results show that the C‐domain of calreticulin is susceptible to proteolytic cleavage and that the N‐ and P‐domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N‐domain, and the third cysteine was found in the reduced form. No post‐translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C‐terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.
The structure of the lipopolysaccharide O‐antigen produced by Flavobacterium psychrophilum (259‐93)
Authors: MacLean Leann L. , Vinogradov Evgeny , Crump Elizabeth M. , Perry Malcolm B. , Kay William W. 
Published: March 2003

Flavobacterium psychrophilum, a Gram‐negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids. In humans Flavobacterium spp. have been associated with neonatal meningitis and septicemia, catheter‐associated bacteremia, and pneumonia. Recently, several F. psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules. Studies on the LPS produced by the bacterium are reported herein. The structure of the antigenic O‐polysaccharide contained in the LPS of F. psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of l‐rhamnose (l‐Rhap), 2‐acetamido‐2‐deoxy‐l‐fucose (l‐FucpNAc) and 2‐acetamido‐4‐((3S,5S)‐3,5‐dihydroxyhexanamido)‐2,4‐dideoxy‐d‐quinovose (d‐Quip2NAc4NR, 2‐N‐acetyl‐4‐N‐((3S,5S)‐3,5‐dihydroxyhexanoyl)‐d‐bacillosamine) (1 : 1 : 1) and having the structure: →4)‐α‐l‐FucpNAc‐(1→3)‐α‐d‐Quip2NAc4NR‐(1→2)‐  α‐l‐Rhap‐(1→ where R is (3S,5S)‐CH3CH(OH)CH2CH(OH)CH2CO‐.
Generation and epitope mapping of high‐affinity scFv to eukaryotic elongation factor 1A by dual application of phage display
Authors: Kjær Svend , Wind Troels , Ravn Peter , Østergaard Morten , Clark Brian F. C. , Nissim Ahuva 
Published: March 2003

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity‐improve and map epitopes of two previously isolated, low‐affinity single‐chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A‐1 and eEF1A‐2. By PCR‐based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage‐based affinity‐selection, two ‘families’ of affinity‐improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nm to eEF1A‐1. Interestingly, two affinity‐improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen‐binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413–450 of eEF1A‐1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage‐antibody repertoire derived scFv were used to select phage‐displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414–418) sequence found in hEF1A‐1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF‐7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.
S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A
Authors: Inoue Akira , Omori Akira , Ichinose Sachiyo , Takahashi Kenichi P. , Kinoshita Yosihiro , Mita Shiro 
Published: March 2003

S1 proteins A–D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross‐linking. In hnRNPs, protein D2 occurred at nuclease‐hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif‐binding factor A (CBF‐A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF‐A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post‐transcriptional processes in gene expression.
CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro
Authors: Cauet Gilles , Balbuena David , Achstetter Tilman , Dumas Bruno 
Published: March 2003

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P‐450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011β) enzyme, which performs the 11β hydroxylation of steroids such as 11‐deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P‐450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P‐450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11β hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, minerallocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.
Oxidized low density lipoproteins downregulate LPS‐induced platelet‐activating factor receptor expression in human monocyte‐derived macrophages
Authors: Hourton Delphine , Stengel Dominique , Chapman M. John , Ninio Ewa 
Published: March 2003

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet‐activating‐factor (PAF). We investigated the effect of oxidized low‐density lipoprotein (oxLDL) on lipopolysaccharide (LPS)‐induced PAF receptor (PAF‐R) expression in human macrophages and the implication of the nuclear factor (NF)‐κB in this regulation. LPS‐treatment (1 µg·mL−1) of macrophages increased PAF binding and PAF‐R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low‐density lipoprotein [ox24hLDL; 100 µg·mL−1; thiobarbituric acid reacting substances: 31 ± 3 nmol equiv. malondialdehyde (MDA)·mg protein LDL−1] diminished PAF‐R expression (−69%; P < 0.05) and mRNA level (− 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF‐κB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS‐induced binding of NF‐κB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 µm), a major component of oxLDL, inhibited the LPS‐induced NF‐κB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF‐R expression in human macrophages by inhibiting LPS‐induced NF‐κB binding to DNA.