bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolism of tRNA in near‐ultraviolet‐illuminated Escherichia coli The tRNA repair hypothesis
Authors: BLANCHETOT Alain , HAJNSDORF Eliane , FAVRE Alain 
Published: March 2005

The relA+‐dependent stringent response is an important component of the mechanism of the near‐ultraviolet‐induced growth delay. However, the behaviour of the intracellular level of ppGpp is unexpected [Thomas et al. (1981) Eur. J. Biochem. 118, 381–387] and this led us to examine the metabolism of tRNAs during the illumination period and the growth lag that follows. Analysis of the gel electrophoresis migration profiles of tRNA molecules, synthesized prior to the illumination period, provides no evidence for tRNA degradation. Rather, it is suggestive of the rearrangement of some cross‐linked tRNA species during the growth lag. By the same technique the neosynthesis of one or several tRNA species escaping the stringent response could be ruled out at the beginning of the growth lag. The behaviour of the cross‐linked tRNAs was followed by a chromatographic procedure allowing the quantitative evaluation of the 8–13 link present in vivo. Upon illumination of growing cells, one observes an initial linear increase of the 8–13 link content. Unexpectedly this is followed during the illumination period by an abrupt decrease. The 8–13 link content then remains stable. The data above suggest that part of the 8–13 link (25–40 %) is eliminated from tRNA without degradation of the molecules involved.A tRNA repair hypothesis is proposed: elimination of the 8–13 link would occur by scission of the N1–C1′ glycosidic bonds at positions 8 and 13 of tRNA. It would be followed by reinsertion of uracil and cytosine in their respective positions.
Inhibition of cell proliferation by interferons
Authors: MOORE Graham , GEWERT Dirk R. , CLEMENS Michael J. 
Published: March 2005

The inhibition of proliferation of Daudi cells in culture by human interferons is characterized by a change in the kinetics of labelling of different size classes of newly synthesized DNA. Initially, labelled precursors are incorporated exclusively into small DNA (Okazaki fragments) in both control and interferon‐treated cells, as revealed by alkaline sucrose gradient centrifugation. In the interferon‐treated cells, there is enhanced labelling of this small DNA after short periods of incorporation and slower conversion to larger DNA size classes, in comparison with the DNA of control cells. This effect is apparent after 12 h of interferon treatment, coincident with the onset of the inhibition of cell proliferation. It becomes progressively more marked up to 4 days, by which time cell growth has ceased completely.Experiments using bromodeoxyuridine as a density label and analysis of radioactive DNA on caesium chloride/caesium sulphate gradients also reveal that some newly replicated DNA may be unstable and may turn over within a few hours of its synthesis. The label derived from DNA breakdown is efficiently reincorporated into newly synthesized molecules. It is suggested that interferon treatment inhibits DNA replication by activating DNA turnover rather than by directly inhibiting synthesis. This effect, together with the progressive retardation of conversion of Okazaki fragments to larger DNA, may lead to the eventual failure of cell proliferation.
The importance of monopole‐monopole and monopole‐dipole interactions on the binding of NADPH and NADPH analogues to p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: WIJNANDS Robert A. , VAN DER ZEE Jolanda , VAN LEEUWEN Johan W. , VAN BERKEL Willem J. H. , MÜLLER Franz 
Published: March 2005

NADPH binding to p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02–0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values < 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate.The experimental data can be explained by a theoretical model in which monopole‐monopole or monopole‐dipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.
Topological location of diamine oxidase in the transverse plane of rabbit liver microsomal membranes
Authors: ARGENTO‐CERÙ Maria Paola , BARGAGLI Anna Maria , SARTORI Claudia 
Published: March 2005

The transverse topology of diamine oxidase within rabbit liver microsomal membranes was studied by examining the proteolytic digestion of sealed or detergent‐permeabilized microsomal vesicles. Trypsin and pronase had no effect on diamine oxidase activity in any incubation conditions tested, while nagarse treatment reduced by 60–70% the enzymic activity of intact microsomes; no further loss of activity was observed in the presence of detergent.These results demonstrate that the active site of diamine oxidase faces the cytoplasmic membrane surface, and suggest that it does not possess or expose on either membrane surfaces bonds susceptible to the proteolytic attack by trypsin or pronase. The possible significance and the biological implications of the results are discussed.
Pyridine‐nucleotide oxidation, Ca2+cycling and membrane damage duringtert‐butyl hydroperoxide metabolism by rat‐liver mitochondria
Authors: BELLOMO Giorgio , MARTINO Anna , RICHELMI Plinio , MOORE Gregory A. , JEWELL Sarah A. , ORRENIUS Sten 
Published: March 2005

Astert‐butyl hydroperoxide is metabolized by the glutathione peroxidase–glutathione reductase enzyme system present in liver mitochondria, rapid and extensive oxidation of NADH and slow NADH oxidation are observed. This NAD(P)H oxidation can be prevented, or reversed, more effectively by 2‐hydroxybutyrate than by isocitrate, indicating an important role of mitochondrial NAD(P)+transhydrogenase activity in maintaining a high NADPH/NADP+ratio for glutathione reductase.In Ca2+‐loaded mitochondriatert‐butyl hydroperoxide‐induced NAD(P)H oxidation is followed by Ca2+release from the mitochondria. If either 2‐hydroxybutyrate or isocitrate is present, no Ca2+release can be induced by the hydroperoxide. Following Ca2+efflux the NAD(P)H oxidation process becomes irreversible and membrane damage occurs. These late effects do not take place if ruthenium red is added to prevent re‐uptake of released Ca2+by the mitochondria. Thus, we conclude that the metabolism oftert‐butyl hydroperoxide leads to a release of mitochondrial Ca2+via oxidation of pyridine nucleotides, and that subsequent membrane damage is not directly associated with this Ca2+efflux but results from continued cycling of released Ca2+.
Catalytical mechanism of the phenylalanyl‐tRNA synthetase from yeast
Authors: THIEBE Rainer 
Published: March 2005

Phenylalanyl‐tRNA synthetase catalyses an AMP‐ATP exchange under conditions where no aminoacylation of tRNA occurs. A plausible explanation for this reaction had not been given so far. The results of the present investigation provide evidence for the following interpretation.tRNAphe induces a polarisation of the ATP in complex with the enzyme; this stimulates (a) the formation of phenylalanyl‐adenylate in the presence of phenylalanine, (b) the hydrolysis of ATP in the absence of phenylalanine and AMP and (c) the transfer of diphosphoryl onto AMP in the presence of AMP, especially when phenylalanine is absent.
Affinity chromatography, on fructose‐bisphosphatase–Sepharose, of two chloroplastic thioredoxins F
Authors: BUC Jean , RIVIÈRE Mireille , GONTERO Brigitte , SAUVE Paul , MEUNIER Jean‐Claude , RICARD Jacques 
Published: March 2005

A new method of purification of chloroplastic thioredoxins has been presented. This method is based on affinity chromatography on fructose‐bisphosphatase—Sepharose columns.Two thioredoxin, fA and fB, may be extracted and purified to homogeneity from the same leaf extract. Whereas fA is monomeric and has an Mr of 11400 ± 500, fB is dimeric with an Mr of 18000 ± 600. The dimer dissociates in two halves in the ultracentrifuge under the effect of high pressures. Raising the ionic strength results in the same effect.Thioredoxins fA and fB activate to similar extents chloroplastic fructose bisphosphatase and NADP–malate dehydrogenase. Chloroplastic sedoheptulose bisphosphatase is activated by thioredoxin fB but not by thioredoxin fA.
Chemical behaviour of cytidine 5′‐monophospho‐N‐acetyl‐β‐d‐neuraminic acid under neutral and alkaline conditions
Authors: BEAU Jean‐Marie , SCHAUER Roland , HAVERKAMP Johan , KAMERLING Johannis P. , BORLAND Lambertus , VLIEGENTHART Johannes F. G. 
Published: March 2005

The chemical behaviour of CMP‐N‐acetylneuraminic acid under neutral and different alkaline conditions has been investigated. The products formed were isolated by ion‐exchange chromatography and gel filtration and analysed by colorimetric methods, thin‐layer chromatography, combined gas‐liquid chromatography/mass spectrometry and/or 360‐MHz 1H‐NMR spectroscopy. A maximum stability of CMP‐N‐acetylneuraminic acid was observed at pH 8–11. In the tested pH range of 6–13, CMP and N‐acetylneuraminic acid were formed in variable amounts as decomposition products. 2‐Deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid was produced at pH > 7; the amount of this substance increased with increasing pH. In anhydrous triethylamine its yield was 50%. A new neuraminic acid derivative, N‐acetyl‐β‐d‐neuraminic acid 2‐phosphate, could be isolated from the mixture of alkaline decomposition products of CMP‐N‐acetylneuraminic acid. The yield of this compound was maximum 22% in anhydrous triethylamine.Because 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid was formed under simulated physiological conditions, it is assumed that this compound, which occurs in tissues and fluids of man and animals, is derived from CMP‐N‐acetylneuraminic acid non‐enzymically also under conditions in vivo.
Interaction of the HB protein of Bacillus globigii with nucleic acids
Authors: WATANABE Fumiyuki , STANKOWSKI Stefan , SCHWARZ Gerhard 
Published: March 2005

Fluorescence of the phenylalanine residues of the HB protein of Bacillus globigii was found to be quenched upon binding to nucleic acids. Using this phenomenon, the binding properties were investigated on the basis of an approach published recently.The stoichiometric numbers were about 10 basepairs per bound protein molecule for double‐stranded DNA and about 10 bases for single‐stranded polynucleotides, independent of salt concentration. Cooperativity parameters were in the range of 50–250. Binding constants were about 5 × 106M−1 (at 0.1 M NaCl) and decreased with increasing salt concentration. From the salt dependence it is inferred that about one Na+ ion is displaced upon binding of a protein molecule to DNA. Therefore the binding site should contain one positively charged amino acid residue.The protein was found to bind with comparable strength to double‐stranded and single‐stranded DNA as well as to poly(rA). Hence it is concluded that the HB protein does not belong to the category of ‘melting proteins’.
Rat‐liver lysosomal sialidase
Authors: MIYAGI Taeko , TSUIKI Shigeru 
Published: March 2005

Purified liver lysosomes, prepared from rats previously injected with Triton WR‐1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity.The intralysosomal sialidase was solubilized from the mitochondrial‐lysosomal fraction of rat liver and partially purified by Sephadex G‐200, or Sephadex G‐200 followed by CM‐cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60000 ± 5000 by gel filtration; if hydrolyzed a variety of sialooligosaccharides, those containing (α2 → 3)sialyl linkages being better substrates than those with (α2 → 6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and fransferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin.In contrast to the intralysosomal silaidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE‐cellulose and CM‐cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maxitoally active at pH 5.8 and had a relative molecular mass of approximately 60000. It also hydrolyzed gangliosides but not submaxillary mucin.
Processing of the 3′ sequence extensions upon the 5S rRNA of a mutant yeast in Xenopus laevis germinal vesicle extract
Authors: PIPER Peter W. , PATEL Nita , LOCKHEART Alan 
Published: March 2005

A processing endonuclease acts to remove a short sequence from the 3′ end of transcripts of the Saccharomyces cerevisiae 5S ribosomal RNA gene in generating the mature sequence of 5S RNA. Cells bearing the nuclear mutation rna82.1 lack this activity and accumulate 5S forms with additional nucleotides at their 3′ termini. 5S RNAs labelled during short pulse‐labellings of the mutant are essentially primary transcripts that mostly have the sequence U‐U‐A‐U‐U‐U‐C[U‐U‐U‐U(U‐U)] added to the 3′ end of normal yeast 5S RNA. They are subjected in vivo to a series of slow processing events whereby this sequence is ultimately replaced by: U‐U(A)1–9 in a substantial proportion of the 5S RNA molecules of the mutant [Piper, P. W., Bellatin, J. A. and Lockheart, A. (1983) EMBO J. 2, 353–359].In higher eukaryotes no endonuclease cleavage occurs during 5S RNA maturation, yet processing at the 3′ ends of certain transcripts made by RNA polymerase III, most notably transfer RNA precursors, is still important. Since the enzymes involved in this processing have not been well characterised, we investigated how the additional sequences upon rna82.1 yeast 5S RNA are processed in vitro in a system from a higher eukaryote that is often used for studying transcription by RNA polymerase III, the Xenopus laevis germinal vesicle extract. Our results are consistent with slow digestion of these 5S molecules by a 3′→ 5′ exonuclease until they become 122–123 nucleotides in length, whereupon digestion ceases. This activity probably participates in the processing of certain Xenopus RNA polymerase III transcripts.
Dissociation of ribulose‐1,5‐bisphosphate carboxylase/oxygenase from spinach by urea
Authors: VOORDOUW Gerrit , van der VIES Saskia M. , BOUWMEISTER Peter Pan 
Published: March 2005

The dissociation of d‐ribulose‐1,5‐bisphosphate carboxylase/oxygenase from spinach, which consists of eight large subunits (L, 53 kDa) and eight small subunits (S, 14 kDa) and thus has a quarternary structure L8S8, has been investigated using a variety of physical techniques.

Gel chromatography using Sephadex G‐100 indicates the quantitative dissociation of the small subunit S from the complex at 3–4 M urea (50 mM Tris/Cl pH 8.0, 0.5 mM EDTA, 1 mM dithiothreitol and 5 mM 2‐mercaptoethanol). The dissociated S is monomeric.
Analytical ultracentrifuge studies show that the core of large subunits, L, remaining at 3–4 M urea sediments with s20,w, = 15.0 S, whereas the intact enzyme (L8S8) sediments with s20,w= 17.7 S. The observed value is consistent with a quarternary structure L8. The dissociation reaction in 3–4 M urea can thus be represented by L8S8→ L8+ 8 S. At urea concentrations c > 5 M the L8 core dissociates into monomeric, unfolded large subunits.
A large decrease in fluorescence emission intensity accompanies the dissociation of the small subunit S. This change is completed at 4 M urea. No changes are observed upon dissociating the L8 core. The kinetics of dissociation of the small subunit, as monitored by fluorescence spectroscopy, closely follow the kinetics of loss of carboxylase activity of the enzyme.
Studies of the circular dichroism of d‐ribulose‐1,5‐bisphosphate carboxylase in the wavelength region 200–260 nm indicate two conformational transitions. The first one ([θ]220 from ‐8000 to ‐3500 deg cm2 dmol−1) is completed at 4 M urea and corresponds to the dissociation of the small subunit and coupled conformational changes. The second one ([θ]220 from ‐3500 to ‐1200 deg cm2 dmol−1) is completed at 6 M urea and reflects the dissociation and unfolding of large subunits from the core. The effect of activation of the enzyme by addition of MgCl2 (10 mM) and NaHCO3 (10 mM) on these conformational transitions was investigated. The first conformational transition is then shifted to higher urea concentrations: a single transition ([θ]220 from ‐8000 to ‐1200 deg cm2 dmol−1) is observed for the activated enzyme.
From the urea dissociation experiments we conclude that both large (L) and small (S) subunits are important for carboxylase activity of spinach d‐ribulose‐1,5‐bisphosphate carboxylase: the L‐S subunit interactions tighten upon activation and dissociation of S leads to a coupled, proportional loss of enzyme activity.
Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML308
Authors: BORTHWICK Andrew C. , HOLMS W. Henry , NIMMO Hugh G. 
Published: March 2005

In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Microbiol. 87, 37–51].
The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion‐red‐Sepharose, from which the enzyme was specifically eluted with NADP+.
Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion‐red‐Sepharose and was essentially inactive; this form could be resolved from the active form by non‐denaturing gel electrophoresis. The other form was specifically eluted from procion‐red‐Sepharose and was partially active; analysis of this form by non‐denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms.
The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one‐dimensional peptide mapping.
Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non‐denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two‐dimensional peptide mapping showed that both forms were dimers of identical subunits.
The active form of the enzyme contained no detectable alkali‐labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount.
The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal).
The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.
Purification and initial characterization of a protein which facilitates assembly of nucleosome‐like structure from mammalian cells
Authors: ISHIMI Yukio , HIROSUMI Jiro , SATO Wakao , SUGASAWA Kaoru , YOKOTA Shoji , HANAOKA Fumio , YAMADA Masa‐atsu 
Published: March 2005

A protein, which facilitates assembly of a nucleosome‐like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735–744]. The protein has been purified to approximately 80% from FM3A cells by using histone‐Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53‐kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53‐kDa peptide bound to core histones and formed a 12‐S complex. This complex contained stoichiometrical amounts of the 53‐kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B. H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12‐S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53‐kDa peptide, formation of a 10.5‐S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53‐kDa peptide was detected in the complex.From above results it is suggested that the 53‐kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53‐kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53‐kDa peptide and nucleoplasmin is discussed.
Platelet aggregation: its relation with ADP‐induced fibrinogen binding to platelets and ADP‐related membrane enzyme activities
Authors: LEGRAND Chantal , DUBERNARD Véronique , CAEN Jacques 
Published: March 2005

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen‐binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto‐ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP‐induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P < 0.01) was found between the extent of platelet aggregation and the amount of 125I‐fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP‐induced platelet aggregation and fibrinogen binding.Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP‐induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP‐induced platelet aggregation.