bluuebunny/crossref_metadata_2025 huggingface dataset:
De novo Sulfation of l‐Tyrosine in HepG2 Human Hepatoma Cells and Its Possible Functional Implication
Authors: Sakakibara Yoichi , Suiko Masahito , Liu Ming‐Cheh
Published: March 2005

HepG2 human hepatoma cells, labeled with [35S]sulfate in the presence of 10–30 μg/ml of cycloheximide, released up to 64% of the amount of free tyrosine‐O ‐[35S] sulfate produced and released by cells labeled in the absence of cycloheximide. A time‐course study revealed that, in cells incubated in medium containing [3H] tyrosine, free [3H] tyrosine‐O ‐sulfate was produced within 5 min of incubation, whereas no [3H] tyrosine‐sulfated proteins were detected until 20 min after the incubation had begun. Using 3′‐phosphoadenosine, 5′‐phospho[35S]sulfate as the sulfate donor, HepG2 cell homogenate was shown to contain enzymic activity catalyzing the sulfation of l‐tyrosine with the formation of tyrosine‐O ‐[35S] sulfate. Upon subcellular fractionation, the majority of the enzyme activity was found in the cytosolic fraction. The enzyme, designated tyrosine sulfotransferase, displayed the optimum activity at pH 8.0 in the presence of 10m Mn2+. Under optimum conditions, the apparent Km of the enzyme for l‐tyrosine, at 4.5‐μM concentration of 3′‐phosphoade‐nosine, 5′‐phosphosulfate, was determined to be 1.95 mM, while that for 3′‐phosphoadenosine, 5′‐phosphosulfate, at 1 mM l‐tyrosine concentration, was 8.3 μM. The Vmax determined under these conditions was 1.05 pmol · min−1. mg protein−1. A tyrosine‐dependence study showed that, for cells labeled with [35S] sulfate, the production and release of free tyrosine‐O ‐[35S] sulfate appeared to proceed actively and increase proportionally to the l‐tyrosine concentration when it was raised above a threshold level in the culture medium. These results may imply a possible involvement of sulfation in removing excess intracellular l‐tyrosine.
Probing the Structure of Hirudin from Hirudinaria manillensis by Limited Proteolysis
Authors: Vindigni Alessandro , De Filippis Vincenzo , Zanotti Giuseppe , Visco Carlo , Orsini Gaetano , Fontana Angelo
Published: March 2005

Hirudin is the most potent and specific inhibitor of the blood‐clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64‐amino‐acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41–49 of the chain. The N‐terminal fragments 1–41 and 1–47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three‐dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N‐terminal well‐structured domain (core) and a C‐terminal flexible polypeptide segment. Determination of the accessible surface area of the three‐dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.
Promoter Function of the Human Glucose‐6‐Phosphate Dehydrogenase Gene Depends on Two GC Boxes that are Cell Specifically Controlled
Authors: Philippe Marianne , Larondelle Yvan , Lemaigre Frédéric , Mariamé Bernard , Delhez Huguette , Mason Philip , Luzzatto Lucio , Rousseau Guy G.
Published: March 2005

Human glucose‐6‐phosphate dehydrogenase is expressed in all cells by a housekeeping gene whose regulatory 5′‐flanking sequence includes at least nine GC boxes. By transient transfection of HeLa and HepG2 cells with constructs containing glucose‐6‐phosphate dehydrogenase gene regions linked to a reporter gene, we have now delineated the core promoter and have located upstream stimulatory and inhibitory sequences. By mutational analysis, we demonstrate that the activity of the core promoter requires two out of seven GC boxes. We show that stimulatory protein 1 (Sp1)‐related factors and activator protein 2 (AP‐2)‐related proteins bind to these two boxes in band‐shift experiments. One point mutation that affects the binding of only the Sp1‐related factors to one or both boxes causes a marked decrease of promoter activity in HepG2 cells but not in HeLa cells. We conclude that (a) two out of many seemingly redundant GC boxes are necessary to drive a Grich housekeeping promoter; (b) factors that bind to GC boxes may exert cell‐type‐specific regulation of housekeeping gene promoter activity; (c) point mutations in the promoter of the glucose‐6‐phos‐phate dehydrogenase gene can inhibit its transcription.
Biochemical Characterization of Human PC‐1, an Enzyme Possessing Alkaline Phosphodiesterase I and Nucleotide Pyrophosphatase Activities
Authors: Belli Sabina I. , Goding James W.
Published: March 2005

PC‐1 is an ecto‐enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC‐1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC‐1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC‐1. The first methionine residue is therefore the initiator. Human PC‐1 is capable of autophosphorylation, and conditions are described in which PC‐1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC‐1 is approximately 10 kDa larger than that of the mouse form. Pulse‐chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N‐linked oligosaccharides. The polypeptide backbone of human PC‐1 is 20 amino acids longer than that of the mouse PC‐1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC‐1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain.
The NADPH: Sulfite Reductase of Escherichia coli is a Paraquat Reductase
Authors: Gaudu Philippe , Fontecave Marc
Published: March 2005

The NADPH:sulfite reductase of Escherichia coli is a soluble enzyme that has a subunit structure α8β4, where the α subunit is a flavoprotein and the β subunit is a metalloprotein. Overexpression of the holoenzyme in E. coli has greatly simplified the purification of this enzyme. Under aerobic conditions, recombinant sulfite reductase catalyzes the reduction of 1,1′‐dimethyl‐4,4′‐bipyridinium dichloride (paraquat) by NADPH, with Km values for paraquat and NADPH of approximately 70 μM and 80 μM, respectively. Since pure flavoprotein α subunit, encoded by the cysJ gene, has similar catalytic activities, it is suggested that paraquat receives electrons directly from the α subunit. A mutant strain lacking an active cysJ gene is resistant to paraquat. The NADPH rferredoxin reductase of E. coli is also a paraquat reductase but with much higher Km values for paraquat and lower enzyme activities. These results suggest that the sulfite reductase is a major paraquat reductase in E. coli and is responsible for the toxic activation of the drug.
Interdomain Loops and Conformational Changes of Glutamate Synthase as Detected by Limited Proteolysis
Authors: Vanoni Maria A. , Mazzoni Alessandra , Fumagalli Prisca , Negri Armando , Zanetti Giuliana , Curti Bruno
Published: March 2005

Azospirillum brasilense glutamate synthase, a complex iron‐sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time‐dependent degradation of glutamate synthase α and β subunits, to yield several fragments of different stability, was observed, the a subunit being more sensitive than the β to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N‐terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the α subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099–3106]. Therefore, these protease‐sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C‐terminal region of glutamate synthase β subunit from tryptic cleavage, supporting the proposal that it contains the pyridinenucleotide‐binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog l‐methionine sulfone, which binds presumably to the N‐terminal region of the a subunit, altered the sensitivity to proteolysis of the sites of the a subunit proposed to be part of links between domains of glutamate synthase. These results show that long‐range conformational changes of glutamate synthase occur on binding of its substrates.

The study of several NADPH‐dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH‐dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.
A Unified Pathway for the Degradation of Ornithine Decarboxylase in Reticulocyte Lysate Requires Interaction with the Polyamine‐Induced Protein, Ornithine Decarboxylase Antizyme
Authors: Mamroud‐Kidron Emanuelle , Omer‐Itsicovich Merav , Bercovich Zippy , Tobias Karin E. , Rom Eran , Kahana Chaim
Published: March 2005

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC‐Az), a polyamineinduced protein. However, the mechanism whereby ODC‐Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte‐lysate–based synthesis and degradation systems, we demonstrate that interaction of ODC‐Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC‐Az binding site of ODC and the ODC binding site of ODC‐Az, acceleration in ODC degradation also requires the previously characterized carboxyl‐terminal destabilizing segment of ODC and a specific segment of ODC‐Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC‐Az binding site is stable under basal degradation conditions. This, together with the ability of anti‐(ODC‐Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC‐Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.
Sequence‐Specific Recognition of a Repetitive DNA Element by a C2H2 Zinc‐Finger Protein in Xenopus
Authors: Schäfer Ute , Rausch Oliver , Bouwmeester Tewis , Pieler Tomas
Published: March 2005

XFG 20–1 is one of several hundred C2H2‐type zinc finger proteins of unknown biological function in Xenopus. Affinity selection of genomic DNA fragments identifies a 54‐bp consensus sequence with high affinity for the XFG 20–1 protein. This sequence is part of a conserved inverted repeat in REM‐1, a 0.5‐kb repetitive, dispersed genomic DNA element. This finding indicates a possible link between the non‐conserved nature of a large subgroup of DNA‐binding C2H2 zinc finger proteins and the non‐conserved nature of putative genomic target sequences. The 19 zinc finger modules of XFG 20–1 are organized in three structurally distinct groups. Of these, only the first and third ones are required and are sufficient for high‐affinity DNA binding. The finding that removal of five internal zinc finger units does not alter the DNase I protection pattern of the complex formed is indicative of a high degree of structural flexibility for linear arrays of zinc fingers, which we imagine to adopt a more rigid structure only upon interaction with their nucleic acid partner molecules.
Primary Structure and Inhibitory Properties of a Subtilisin‐Chymotrypsin Inhibitor from Streptomyces Virginiae
Authors: Terabe Mahito , Kojima Shuichi , Taguchi Seiichi , Momose Haruo , Miura Kin‐ichiro
Published: March 2005

A novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin‐inhibitor‐like (SIL) inhibitor family by sequence analysis of its ammo‐terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. & Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522–524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward α‐chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI‐family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the β‐sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants K1 obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for α‐chymotrypsin. The P1 site was identified as methionine, which was in good agreement with the substrate specificity of α‐chymotrypsin. SSI, which also possesses a methionine residue at the PI site, inhibits a‐chymotrypsin poorly (inhibitor constant, 4.0 μM). Such a difference in the inhibitory properties of SIL8 and SSI toward α‐chymotrypsin is discussed on the basis of the structures of the inhibitors.
The Folding of the Bifunctional TRP3 Protein in Yeast is Influenced by a Translational Pause which Lies in a Region of Structural Divergence with Escherichia coli Indoleglycerol‐Phosphate Synthase
Authors: Crombie Tanya , Boyle John P. , Coggins John R. , Brown Alistair J. P.
Published: March 2005

The yeast TRP3 gene encodes a bifunctional protein with anthranilate synthase II and indoleglycerol‐phosphate synthase activities. Replacing ten consecutive non‐preferred codons in the indoleglycerol‐phosphate synthase region of the TRP3 gene with synonymous preferred codons (to create the TRP3pr gene; translational pause replaced) causes a 1.5‐fold reduction in relative indoleglycerol‐phosphate synthase activity [Crombie, T., Swaffield, J. C. & Brown, A. J. P. (1992) J. Mol. Biol. 228, 7–12]. Here, we report that both the anthranilate synthase II and indoleglycerol‐phosphate synthase domains are affected to similar extents when the translational pause is removed. Also, structural modelling of the yeast indoleglycerol‐phosphate synthase domain against the X‐ray crystal structure of indoleglycerol‐phosphate synthase from Escherichia coli indicates that the translational pause lies in a region of structural divergence between similar structures. To probe the role of cytoplasmic heat‐shock protein 70 (Hsp70) chaperones in Trp3 protein folding, anthranilate synthase and indoleglycerol‐phosphate synthase activities were measured in ssa and ssb mutants. Neither indoleglycerol‐phosphate synthase nor anthranilate synthase were affected significantly in the ssb mutant. However, depletion of Hsp70 proteins encoded by the SSA genes led to decreased anthranilate synthase and indoleglycerol‐phosphate synthase activities from the TRP3 gene, suggesting that both domains depend to some extent upon the SSA chaperone family. The data are consistent with roles for both the translational pause and Ssa chaperones in Trp3 protein folding in vivo.
Isolation of a Novel Plasmodium falciparum Gene Encoding a Protein Homologous to the Tat‐Binding Protein Family
Authors: Hirtzlin Jocelyne , Färber Petra M. , Franklin Richard M.
Published: March 2005

We have cloned a Plasmodium falciparum gene that belongs to the nuclear Tat‐binding protein (TBP) gene family. This gene, PfTBP, is (A+T)‐rich and encodes a 49.5‐kDa protein. The predicted protein encoded by this gene has highest similarity to the slime mold protein DdTBP10 (86%) and to the yeast protein SUG1 (81.8%), both of which belong to the Tat‐binding protein family. In agreement with the characteristics of this family, PfTBP contains a highly conserved domain of approximately 200 amino acids, in which are found the motifs A and B of ATPases, and amino acid sequences characteristic of a large family of RNA or DNA helicases, suggesting a role in RNA or DNA unwinding. Like DdTBP10, the PfTBP protein has a heptad repeat of four leucine residues, reminiscent of a leucine zipper motif known to mediate dimerization. We have further characterized PfTBP gene expression by Northern‐blot analysis. This gene is expressed in a stage‐specific manner, with higher expression in the late trophozoite stage. The recombinant PFTBP gene has been expressed in Escherichia coli and a polyclonal antiserum has been raised in rabbits against the recombinant protein. This antibody has been used to study the protein in the parasite. The gene product is expressed in a stage‐specific manner with higher expression in the late trophozoite and schizont stages, and is localized in the nucleus of the erythrocytic stage parasite. Thus the protein might have a function in DNA synthesis and/or in transcription, as is the case for other Tat‐binding proteins.
The Reaction of Hydrogen Peroxide with Pulsed Cytochrome bo from Escherichia coli
Authors: Moody A. John , Rich Peter R.
Published: March 2005

The reaction of hydrogen peroxide (H2O2) with pulsed cytochrome bo leads to characteristic spectral changes in the enzyme. The difference spectrum shows minima at 401, 494 and 628 nm, and maxima at 420, approximately 468, 526 and 556 nm. Δɛ420–ɛ401 is in the range 73–86 mM−1· cm−1 and Δɛ556–ɛ628s is 7.7–9.6 mM−1· cm−1 (taking Δɛ560–ɛ580 for the reduced minus oxidised spectrum to be 20.5 mM−1· cm−1). The stoichiometry of the reaction, determined by titration of the spectral changes, is 1:1. The second order rate constant for the reaction, which is 1.0–1.5×103M−1·s−1 at 20°C, is independent of pH over the range 6.5–8.0. The product of the reaction decays with a first‐order rate constant in the range 1–4×10−4 s−1 so the Kd value is apparently in the range 0.05–0.40 μM. The spectral changes observed immediately after quinol‐induced turnover, or during steady‐state turnover induced by hydrazine or by carbon monoxide, are qualitatively the same as those induced by H2O2 though of lower amplitude. H2O2 addition perturbs the hydrazine‐induced or CO‐induced steady states by increasing the amplitude of the spectral changes, but there is no qualitative change. From this observation, and the 1:1 Sociometry of the reaction, we conclude that the intermediate induced by H2O2, which we term F, requires donation of only two electrons to the enzyme from an external source.
Inhibition of Protein Synthesis by the Heme‐Controlled Eif‐2α kinase Leads to the Appearance of mRNA‐Containing 48S Complexes that Contain eIF‐4E but Lack Methionyl‐tRNAf
Authors: Joshi Bhavesh , Morley Simon J. , Rhoads Robert E. , Pain Virginia M.
Published: March 2005

Phosphorylation of the initiation factor eIF‐2 by the heme‐regulated eIF‐2α kinase (HCR) results in pronounced inhibition of protein synthesis and of binding of Met‐tRNAf to 40S subunits in reticulocyte lysates. This inhibition is associated with the appearance of a more rapidly sedimenting 48S complex; this contains mRNA detectable by poly(U) hybridization, but not Met‐tRNAf. In contrast, 48S complexes that accumulate in the presence of the initiation inhibitor edeine contain both Met‐tRNAf and mRNA. We have compared the composition of the particles that accumulate in the presence of HCR with those seen in the presence of edeine and find that both particles contain the cap binding protein, eIF‐4E. Moreover, both particles exhibit a buoyant density of 1.40 g/ml in CsCl equilibrium density gradients. This is consistent with the presence of 500–700 kDa of protein additional to ribosomal structural protein, and suggests the presence of eIF‐3 on both types of 48S complex. Lysates pre‐treated with HCR and then treated with edeine show the ability to accumulate 48S complexes containing Met‐tRNAf, though at a slower rate than control lysates. These observations are discussed in the light of mechanisms previously suggested for the appearance of 48S particles following HCR treatment. In addition, we have observed association of eIF‐4E with polysomes and 80S monosomes in reticulocyte lysates, suggesting that this factor may not be released immediately following the binding of the 40S ribosomal subunit to the 5′ end of the mRNA.
The Structure of the Bovine Cathepsin B Gene
Authors: Mordier Sylvie B. , BÉChet Daniel M. , Roux Marie‐Paule , Obled Alain , Ferrara Marc J.
Published: March 2005

Cathepsin B is a cysteine proteinase which plays an important role in lysosomal proteolysis. We report here the characterization of a 15‐kbp genomic clone of the bovine cathepsin B gene. Bovine preprocathep‐sin B is encoded by nine exons from translational initiation to stop codon. The last exon is sandwiched between Alu‐like short interspersed nuclear elements. Alternate use of polyadenylation sites generates three transcripts encoding bovine cathepsin B. In all tissues tested, 3′ end cleavage was found to occur in equal proportion at the first and second polyadenylation site, producing transcripts of 2.6‐kb. Polyadenylation at the third polyadenylation site generates a 3.2‐kb mRNA, only expressed at low levels and in a developmental and tissue‐specific manner. Due to micro‐heterogeneities between genomic and cDNA clones, sequence polymorphism was investigated in the trailer region. DNA sequencing of PCR‐amplified genomic fragments revealed that, in contrast to the protein‐encoding region, genetic variability exists in the 3′ untranslated region. Polymorphism in the trailer was confirmed in cathepsin B mRNA by ribo‐nuclease‐protection assays. We finally emphasize that while exon‐exon boundaries in mature cathepsin B are well conserved from nematodes to mammals, exons also tend to correspond to discrete units of cathepsin B structure.
Variations in Fluorescence and Enzymic Properties of Bovine Dihydrofolate Reductase • NADPH Complex During the Slow Conformational Change Induced by Coenzyme Binding
Authors: Rimet Odile , Chauvet Michele , Dell'amico Monique , Noat Georges , Bourdeaux Madeleine
Published: March 2005

When NADPH was added in large excess to bovine dihydrofolate reductase (H2folate reductase), there was a slow isomerization process between two conformers of the binary complex (B1⇌ B2), as shown by changes in the fluorescence properties. Thus, we monitored the time dependence of (a) the quenching of protein intrinsic fluorescence intensity, (b) the polarization state of the fluorescence light emitted by NADPH · H2folate reductase complexes and (c), from a more biological point of view, the enzymic activity of binary complex solutions. The kinetics for these three processes were in good agreement using the same temperature conditions. Furthermore, fluorescence studies provided information on the NADPH environment in the binary complex. As soon as NADPH bound to H2folate reductase, light emitted by the invariant Trp24 residue located within the coenzyme‐binding site was quenched by an energy‐transfer process. Moreover, Trp57 and/or Trp113 emissions were partially quenched. The subsequent NADPH‐bound protein conformational change caused an additional quenching, probably of Trp57 and/or Trp113 emissions. Thus, NADPH · H2folate reductase conformation was modified but no change was observed at the coenzyme‐binding site, at least in our fluorescence study. These results were confirmed by polarization measurements. The conformational change, as well as the instantaneous NADPH binding, resulted in a more rigid form of the protein, as shown by an increase in steady‐state anisotropy values. Finally, the isomerization process led to a more active enzymic form.
Molecular Characterisation of the ADP/ATP‐Transporter cDNA from the Human Malaria Parasite Plasmodium Falciparum
Authors: Hatin Isabelle , Jaureguiberry Ginette
Published: March 2005

We have isolated a cDNA sequence encoding the ADP/ATP transporter in Plasmodium falciparum. The sequence analysis revealed an open reading frame encoding 301 amino acids and showed significant similarities to known eukaryotic translocases such as that of Chlorella (up to 67.2% identity) and the human transporter (61.2%). RNA blot analysis showed the presence of mRNA encoding for a 33.7‐kDa ADP/ATP transporter. During the cell cycle of the parasite the expression levels of the transcripts fluctuate. The mitochondrial ADP/ATP transporter could play a role in energy metabolism of P. falciparum and makes this transporter an excellent target for chemotherapy.
The Role of the CANNTG Promoter Element (E box) and the Myocyte‐enhancer‐binding‐factor‐2 (MEF‐2) Site in the Transcriptional Regulation of the Chick Myogenin Gene
Authors: Malik Sundeep , Huang Chang‐Fen , Schmidt Jakob
Published: March 2005

We have carried out an analysis of 833 bp of the chick myogenin gene 5′ flanking sequence. A 131‐bp segment of this upstream region, which contains a CANNTG promoter element (E box) and a myocyte‐enhancer‐binding‐factor‐2 (MEF‐2) site, acts as a full promoter. It resembles the human and the mouse myogenin promoters in the structure and disposition of regulatory elements, including a TATA box and the transcription start site. Examination of eight myogenic factor/E protein combinations co‐transfected with several myogenin promoter constructs into HeLa cells, reveals that the chick myogenin/E2–5 combination maximally activates the myogenin promoter. This activation is mediated through the E box motif; the MEF‐2 site limits the factor combinations that can activate the myogenin promoter and enhances activation by myogenin/E2–5. We have found previously that activation of protein kinase C inactivates the transcription of the chick myogenin gene [Huang, C.‐F., Neville, C. M. & Schmidt, J. (1993) Control of myogenic factor genes by the membrane depolarization/protein kinase C cascade in chick skeletal muscle, FEBS Lett. 319, 21–25]. In this study, we show that the activation of protein kinase C inhibits the factor bound to the E box, which thereupon negatively regulates the activity of the MEF‐2 binding protein.
Transcription of Immune Genes in the Giant Silkmoth, Hyalophora Cecropia, is Augmented by H2O2 and Diminished by Thiol Reagents
Authors: Sun Shao‐Cong , Faye Ingrid
Published: March 2005

Insects have an effective humoral immune system, residing in immune proteins that are synthesized largely in the fatbody. κB‐like motifs upstream of the immune protein genes bind the Cecropia immunore‐sponsive factor (CIF), and confer high levels of inducible expression.

We have extended our studies and sought evidence that oxygen‐derived active species might modulate the expression of the immune protein genes and the activation of CIF after the administration of different inducers. Mitogens, like arachidonic acid, phorbol esters, phytohemagglutinin, bacteria, and components of microbial cell envelopes stimulate expression of the attacin genes, both in vivo and in vitro. A general stimulant of oxidative stress, H2O2, stimulated expression of these genes and the weak immune response to wounding was greatly augmented by administration of H2O2. These responses were largely or entirely inhibited by dithiothreitol and by N‐acetylcysteine. Nonspecific responses were excluded since immune genes failed to respond to albumin or starch, and the expression of a non‐immunoresponsive gene was not affected. Electrophoretic mobility‐shift assays showed that H2O2 and bacteria, when administered in vitro, could activate CIF in fatbody cells and that dithiothreitol and N‐acetylcysteine prevented this process. Our data suggest that the induction of the immune protein genes is mediated through the activation of CIF, contingent upon thiol oxidation induced by oxidative stress.
Regulation of the Interferon‐indueible Protein Kinase PKR and (2′‐5′)oligo(Adenylate) Synthetase by a Catalytically Inactive PKR Mutant Through Competition for Double‐stranded RNA Binding
Authors: Sharp Tyson V. , Xiao Qiurong , Justesen Just , Gewert Dirk R. , Clemens Michael J.
Published: March 2005

The interferon‐indueible double‐stranded RNA‐dependent protein kinase PKR has been suggested to function as a tumour suppressor gene product. Catalytically inactive mutants of PKR give rise to a tumorigenic phenotype when overexpressed in NIH‐3T3 fibroblasts and this has been attributed to a dominant negative effect on the activity of the wild‐type enzyme. Here we show that the mutant with Lys296 replaced by Arg, [K296R]PKR, not only inhibits the protein kinase activity of wild‐type PKR but is also inhibitory towards another double‐stranded RNA‐dependent enzyme, the 40‐kDa form of (2′‐5′)oligo(adenylate) synthetase. Inhibition of both wild‐type PKR and (2′‐5′)oligo(adenylate) synthetase is reversed by adding higher concentrations of double‐stranded RNA. These results suggest competition between [K296R]PKR and wild‐type PKR or (2′‐5′)oligo(adenylate) synthetase for limiting amounts of double‐stranded RNA. Moreover, the data imply that the tumorigenic effect of this PKR mutant could be due to inhibition of additional pathways requiring low levels of double‐stranded RNA for activation and cannot be unambiguously attributed to inhibition of endogenous PKR itself.
2,3–Dihydroxybenzoic Acid Decarboxylase from Aspergillus niger
Authors: Santha Ramakrishnan , Savithri Handanahal S. , Rao N. Appaji , Vaidyanathan Chelakara S.
Published: March 2005

2,3‐Dihydroxybenzoic acid decarboxylase, the last enzyme in the fungal metabolism of indole to catechol, catalyzes the non‐oxidative decarboxylation of 2,3‐dihydroxybenzoic acid to catechol. Unlike most other decarboxylases, this enzyme does not require a cofactor, underlining the importance of active‐site residues in the reaction mechanism. Earlier studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidyanathan, C. S. (1989) Biochem. Biophys. Res. Commun. 165, 20–26], have shown that the sulfhydryl agent N‐ethylmaleimide (MalN Et) inactivated the enzyme by modifying a single class of cysteine residues and that this inactivation was prevented in the presence of salicylate, a substrate analogue. In the present study, this essential cysteine residue has been identified by specific labelling with [14C]‐MalN Et using the differential labelling technique. The stoichiometry of incorporation of [14C]MalN Et was approximately one/subunit of the homotetrameric protein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequent reverse‐phase chromatography of the peptide mixture. The sequence of the major radioactive peptide that was identified to be the active‐site peptide, was LLGLAETCK. A search for sequences similar to this active‐site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constructed from the sequences of peptides derived from enzymic cleavage of the protein using endoproteinase Glu‐C and trypsin did not share any significant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first report on the active‐site peptide and the partial primary structure of a non‐oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus.
Immunochemical Determination of Human Cholesterol 7α‐Hydroxylase
Authors: Maeda Yo‐rio , Eggertsen Gösta , Nyberg Björn , Setoguchi Toshiaki , Okuda Kyu‐Ichiro , Einarsson Kurt , Björkhem Ingemar
Published: March 2005

To produce antibodies for determination of the protein mass of human cholesterol 7α‐hydroxylase, a fusion protein was prepared from an in‐frame fusion gene containing the cDNA for human cholesterol 7α‐hydroxylase near the 3′ terminus of the lacZ gene of Escherichia coli. The fusion protein was purified by (NH4)2SO4 fractionation and gel‐filtration chromatography on a Sephacryl column. Rabbits were immunized with this fusion protein and antisera were obtained.

IgG was prepared by submitting antisera to chromatography on protein‐A‐Sepharose. Antibodies directed against bacterial proteins including β‐galactosidase were removed by affinity chromatography on a column to which bacterial proteins of E. coli containing β‐galactosidase had been immobilized. Evidence that the antibodies are indeed reactive against human liver cholesterol 7α‐hydroxylase was obtained by immunoblot analysis with human cholesterol 7α‐hydroxylase expressed in COS cells from the coding region of the human cholesterol 7α‐hydroxylase cDNA.

The antiserum inhibited the activity of cholesterol 7α‐hydroxylase in human liver microsomes by approximately 70%. On immunoblotting of solubilized human liver microsomes, a positive band was obtained at a position corresponding to the protein mass for human cholesterol 7α‐hydroxylase. When calibration was performed using the fusion protein, a linear relationship was observed between the density and the amount of protein. Proportionality was also observed between the density and the amount of protein for microsomes of COS cells transfected with the coding region of the human cholesterol 7α‐hydroxylase cDNA.

Liver microsomes from patients treated with cholestyramine (n= 3) were shown to contain levels of cholesterol 7α‐hydroxylase protein approximately twofold higher than those of liver microsomes from untreated patients (n= 6; P < 0.02), whereas cholesterol 7α‐hydroxylase activity was approximately sixfold higher in liver microsomes from the cholestyramine‐treated patients than in the corresponding preparations from the untreated patients (P <0.02). The higher activities observed in cholestyramine‐treated patients, therefore, cannot be explained only by an increased amount of protein, suggesting a posttransla‐tional mechanism to increase the activity of human cholesterol 7α‐hydroxylase in addition to the transcrip‐tional control.
The Oxidation of Methylamine in Paracoccus denitrificans
Authors: De Gier Jan‐Willem L. , Van Der Oost John , Harms Nellie , Stouthamer Adriaan H. , Van Spanning Rob J. M.
Published: March 2005

The in vivo oxidation of methylamine has been studied in Paracoccus denitrificans. Four components are involved in the electron transfer from methylamine to oxygen; methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome‐c oxidase. In P. denitrificans, MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. In the present study, site‐directed mutants have been used to demonstrate participation of cytochrome c550 and the aa3‐type cytochrome‐c oxidase. Moreover, evidence is provided for the operation of alternative routes, branching from amicyanin, in which at least cytochrome c1 and the cbb3‐type cytochrome‐c oxidase are involved.
An NMR Study of the Interaction of Cardiotoxin γ from Naja nigricollis with Perdeuterated Dodecylphosphocholine Micelles
Authors: Dauplais Marc , Neumann Jean Michel , Pinkasfeld Suzanne , Ménez André , Roumestand Christian
Published: March 2005

We investigated the interaction of toxin γ, a cardiotoxin from the venom of the elapid Naja nigricollis, with perdeuterated dodecylphosphocholine (DodPCho) micelles using standard two‐dimensional proton NMR spectroscopy. The proton spectrum resonances of the micelle‐bound toxin γ were assigned, and the chemical shifts of the backbone and side‐chain protons were compared with those determined in the absence of DodPCho. We observed that DodPCho induced large chemical shift changes on residues localized on the hydrophobic face of the toxin. These changes are not associated with conformational changes of the toxin. However, the micellar environment may induce some stabilization of the triple‐stranded β sheet, the major component of the protein structural core. Since the proton NMR spectrum of toxin α, a structurally related neurotoxin extracted from the same venom, was unaffected by the presence of the micelles, we came to the conclusion that the observed effects are specific to cardiotoxins. The present results give direct evidence of the contibution of the hydrophobic face of the toxin to the toxic site and further suggest a possible mechanism of action of eardiotoxin on biological bilayers.
An Agarose‐Based Gel‐Concentration System for Microsequence and Mass Spectrometric Characterization of Proteins Previously Purified in Polyacrylamide Gels Starting at Low Picomole Levels
Authors: Rider Mark H. , Puype Magda , Van Damme Jozef , Gevaert Kris , DE Boeck Stefan , D'Alayer Jacques , Rasmussen Hanna H. , Celis Julio E. , Vandekerckhove Joel
Published: March 2005

An agarose‐based concentration gel system is described for eluting and concentrating proteins previously purified either in one‐dimensional or two‐dimensional gels. Using the technique, proteins can be concentrated from about 1 ml into volumes as small as 10 μl. After the proteins have been melted out of the agarose gels, they can be digested with proteases, producing peptide patterns similar to those observed with in‐solution digestions. The overall peptide recovery, calculated from the amount of protein loaded on the primary separating gel to the collection of fragments after HPLC, is at least 70% of the peptide yields obtained with digests of the same amount of protein in free solution. These results are routinely obtained with 50 pmol amounts (referring to amounts of protein initially loaded on the primary gel). Proteins can also be analysed by a combination of microsequencing and on‐line electrospray mass spec‐trometry, allowing their identification by peptide mass fingerprinting.
Purification and Amino Acid Sequence of Aminopeptidase P from Pig Kidney
Authors: Romero Carlota Vergas , Neudorfer Ingrid , Mann Karlheinz , Schäfer Wolfram
Published: March 2005

Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane‐bound glycoprotein with n‐butanol. A coupled enzymic assay, using Gly‐Pro‐Pro‐NH‐Nap as substrate and dipeptidyl‐peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six TV‐glycosylation sites, all contained in the N‐terminal half of the protein, were characterized.
Basic Fibroblast Growth Factor Modulates the Aminophospholipid Translocase Activity Present in the Plasma Membrane of Bovine Aortic Endothelial Cells
Authors: Julien Michel , Tournier Jean‐Francois , Tocanne Jean‐Francois
Published: March 2005

Vascular endothelial cells form the inner nonthrombogenic lining of the large blood vessel. Through back‐exchange and fluorescence recovery after photobleaching experiments and using the two fluorescent lipids 1 ‐acyl‐2–[6–[N ‐(7‐nitrobenz‐2–oxa‐1,3–díazol‐4‐yl)amino]hexanoyl]glycerophosphocholine and 1–acyl‐2[6‐N ‐(7‐nitrobenz‐2–oxa‐1,3–díazol‐4–yl)amino]hexanoyl]glycerophosphoethanolamine, we have recently shown that an energy‐dependent and protein‐dependent aminophospholipid translocase activity is present in the plasma membrane of cultured bovine aortic endothelial cells, which specifically transports phosphatidylethanolamine from the outer leaflet toward the inner leaflet of the membrane lipid bilayer. In the present study, using the same approach and 1‐acyl‐2‐[6–N ‐(7‐nitrobenz‐2–oxa‐1,3‐díazol‐4‐yl)ami‐no]hexanoyl]glycerophosphoserine as the probe, it is shown that this conclusion is also valid for phosphatidylserine. Furthermore, evidence is presented indicating that this aminophospholipid translocase activity can be maintained, suppressed, and restored at will, depending on the conditions of cell incubation. Thus, the translocase activity is detected for cells maintained in their normal culture medium or in a serum‐free incubation medium [Dulbecco's modified Eagle's medium (DMEM)] supplemented with the basic fibroblast growth factor, whereas inhibition is observed for cells exposed for at least 2 h to DMEM. The translocase activity is restored when these pretreated cells are further incubated at least for 1 h in the presence of serum or of basic fibroblast growth factor. In view of the importance of basic fibroblast growth factor as a mitogenic and differentiating agent for vascular endothelial cells, various growth factors were tested (acidic fibroblast growth factor, epidermal growth factor, platelet‐derived growth factor, transforming growth factors α and β, vascular endothelial growth factor, interferon γ, tumor‐necrosis factor, insulin, and interleukin 4). Only basic fibroblast growth factor was active in the maintenance and restoration of the translocase activity. With respect to the effects of serum, evidence is presented showing that high‐density lipoproteins might play a role in the control of the translocase activity. However, the positive effects of basic fibroblast growth factor, serum and high‐density lipoproteins on the translocase activity were suppressed when experiments were carried out in the presence of an anti‐(basic fibroblast growth factor) IgG, thus indicating that in all cases, basic fibrobiast growth factor was directly involved in the modulation of the aminophospholipid translocase activity present in the plasma membrane of bovine aortic endothelial cells.
Structural Elucidation of the Biological Repeating Unit of O‐specific Polysaccharide from Citrobacter Serotype O41
Authors: Ravenscroft Neil , Dabrowski Janusz , Romanowska Elzbieta
Published: March 2005

Sugar analysis of the O‐specific polysaccharide produced by Citrobacter serotype O41 revealed the presence of a hexasaccharide repeating unit, which includes the unusual 3‐amino‐N ‐(d‐3′hydroxybutyryl)‐3,6‐dideoxy‐d‐galactosyl residue (Fuc3NAcyl). The structure of the repeating unit was determined by extensive use of homo‐ and heteronuclear two‐dimensional NMR spectroscopy, including the application of long‐range 1H‐13C correlation experiments and NOE studies to establish the sequence of sugar units. Identification of the Glcβ1→2Fuc3NAcylβ→6GlcNAcα1→ sequence at the non‐reducing terminus establishes the biological repeating unit of this O‐specific polysaccharide as: →2Glcβ‐2Fuc3NAcyl‐β1→6GlcNAcα1→(Glcα1‐→2)4Galβ1→3GalNAcβ1→. This structure is similar to that found for the O‐specific polysaccharide isolated from Hafnia alvei strain 1211 [Katzenellenbogen, E., Romanowska, E., Dabrowski, U. & Dabrowski, J. (1991) Eur. J. Biochem. 200, 401–407], which differs in having an acetyl substituent at O4 of the Fuc3NAcyl residue and a branch point of GalNAcα substituted by Glcβ at O3; these differences are responsible for the only weak serological cross‐reactivity of the two strains.
Selective Inhibition of p70 S6 Kinase Activation by Phosphatidylinositol 3‐Kinase Inhibitors
Authors: Petritsch Claudia , Woscholski Rüdiger , Edelmann Helga M. L. , Parker Peter J. , Ballou Lisa M.
Published: March 2005

Treatment of fibroblasts with wortmannin or demethoxyviridin, two potent inhibitors of phosphatidylinositol 3‐kinase, prevents the activation of ribosomal protein S6 kinase, which is induced by a variety of external stimuli. Concentrations giving 50% inhibition of 45 nM (wortmannin) and 400 nM (demethoxyviridin) were obtained when epidermal growth factor was used as an S6 kinase activator; with platelet‐derived growth factor, the concentrations giving 50% inhibition were about three‐times higher. Western‐blot analysis and immunocomplex kinase assays showed that wortmannin and demethoxyviridin specifically block the phosphorylation and activation of p70 S6 kinase without affecting the Mr 90000 ribosomal S6 kinase (p90rsk) or mitogen‐activated protein kinases. Consistent with the irreversible nature of the inhibition of phosphatidylinositol 3‐kinase by these compounds, treatment of cells with wortmannin, followed by washing out of the inhibitor, still led to inhibition of p70 S6 kinase activation. Several S6 kinase agonists not previously known to activate phosphatidylinositol 3‐kinase (A23187, bombesin and phorbol 12‐myristate 13‐acetate) were found to increase the production of phosphatidylinositol 3,4,5‐trisphosphate in a wortmannin‐sensitive manner. These results support a model in which phosphatidylinositol 3‐kinase acts upstream of p70 S6 kinase in a mitogenic signalling pathway; the existence of a phosphatidylinositol 3‐kinase‐independent pathway is also evident.