bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction between the Different Domains of Aminoacyl‐tRNA and the Elongation‐Factor‐Tu · Kirromycin Complex
Authors: GUESNET Joëlle , PARLATO Giuseppe , PARMEGGIANI Andrea 
Published: March 2005

In this work, we have studied the effect of aa‐tRNA and derived 3′ aminoacylated fragments on the EF‐Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa‐tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C‐A‐Val and Val‐tRNA1Val 3′ half molecule, as well as a purification method for short 3′ aminoacylated fragments based on the amino group charge, were newly developed for this work. Aminoacyl‐adenosine was found to be able to stimulate the EF‐Tu · kirromycin GTPase, but only to a very small extent. Increasing the length of the aminoacylated fragments increased the stimulatory effect as follow: A‐Val ≪ C‐A‐Val < 3′ valyladenosine dodecanucleotide ≪ Val‐tRNA1Val 3′ half molecule < Val‐tRNA1Val. The presence of ribosomes did not affect the order of effectiveness, but increased the basic GTPase activity of EF‐Tu · kirromycin and the stimulation by aa‐tRNA, its 3′ half molecule and even more by its 3′ short fragments. The effect of aa‐tRNA and derived 3′ fragments in the absence of ribosomes was not influenced by MgCl2 concentrations of 5–30 mM whereas, in the presence of ribosomes, low concentrations of MgCl2 (5 mM) greatly reduced the stimulation of aa‐tRNA and, to a lesser extent, also the effect of the C‐C‐A‐aa as well as the basic activity of the EF‐Tu · kirromycin GTPase.The extent of the stimulation by aa‐tRNA, and even more by C‐C‐A‐aa, depends on the nature of the amino acid. Among the aminoacyl side chains tested (Arg‐, Phe‐, Val‐, Met‐, Leu‐, Lys‐) arginine was found to be the most active and leucine the least.Our results show that (a) the 3′ aminoacylated extremity is of prime importance for the stimulation of the EF‐Tu GTPase, (b) in the 3′ extremity there are critical sequences for the interaction with EF‐Tu and (c) other domains of the aa‐tRNA molecule are capable of influencing this reaction: one of the most important is the region including the TΨC loop and stem.
The Accessibility of the Active Site and Conformation States of the β2 Subunit of Tryptophan Synthase Studied by Fluorescence Quenching
Authors: LANE Andrew N. 
Published: March 2005

The rate of quenching of the fluorescence of pyridoxal 5′‐phosphate in the active site of the β2 subunit of tryptophan synthase from Escherichia coli was measured to estimate the accessibility of the coenzyme to the small molecules iodide and acrylamide. The α subunit and the substrate l‐serine substantially reduced the quenching rate. For iodide, the order of decreasing quenching was: Schiff's base of Nα‐acetyl‐lysine with pyridoxal 5′‐phosphate > holoβ2 subunit > holoα2β2 complex ∼ holoβ2 subunit +l‐serine > holoα2β2 complex +l‐serine. The coenzyme in the β2 subunit is apparently freely accessible to both iodide and acrylamide (k∼× 109 M−1 s−1), but the α subunit and l‐serine decrease the rate by factors of 2–5.Quenching of the fluorescence of the single tryptophan residue of the β2 subunit revealed that the apo and holo forms exist in different states, whereas the α subunit stabilises a third conformation. As the α subunit binds to the β2 subunit, the tryptophan residue, which is within 2.2 nm of the active site of the β2 subunit, probably rotates with respect to the plane of the ring of the coenzyme, such that fluorescence energy transfer from tryptophan to pyridoxal phosphate is greatly reduced.The α subunit strongly protects the active‐site ligand indole propanol phosphate from quenching with acrylamide, consistent with the active site being deep in a cleft in the protein.Iodide induces dissociation of the holoα2β2 complex [E. W. Miles & M. Moriguchi (1977) J. Biol. Chem. 252, 6594–6599]. The effect of iodide on the fluorescence properties of holoα2β2 complex allows us to estimate an upper limit for the dissociation constant for the α2β2 complex of 10‐8M, in the absence of iodide.
9,10‐Dihydrophenanthrenes as Phytoalexins of Orchidaceae
Authors: FRITZEMEIER Karl‐Heinrich , KINDL Helmut 
Published: March 2005

Hydroxy derivatives of 9,10‐dihydrophenanthrenes, orchinol and hircinol, were isolated from bulbs of Orchidaceae which had been induced to accumulate phytoalexins. Incorporation of radioactive precursors, l‐phenylalanine and various hydroxycinnamic acids, has been investigated by feeding experiments in vivo. m‐Coumaric acid and dihydro‐m‐coumaric acid were found to be efficiently incorporated into the dihydrophenanthrene derivatives. Dihydro‐m‐coumaric acid was not only converted into the dihydrophenanthrenes but was also formed from l‐phenylalanine in the same tissue; it was thus proved to be an intermediate.The role of dihydro‐m‐coumaric acid was substantiated by studies in vitro. An active stilbene synthase was detected in enzyme preparations from induced orchid bulbs and assayed with different CoA esters. The enzyme, characterized on the basis of its substrate specificity, selectively converted dihydro‐m‐coumaroyl‐CoA plus malonyl‐CoA into 3,3′,5‐trihydroxybibenzyl.The role of 3,3′,5‐trihydroxybibenzyl as physiological intermediate was further corroborated by investigations with intact plants. Both its formation from phenylpropanoids and its conversion into orchinol was demonstrated.The data provided evidence for a biosynthetic sequence originating from l‐phenylalanine and leading to 9,10‐dihydrophenanthrenes via m‐coumaric acid, dihydro‐m‐coumaric acid, and 3,3′,5‐trihydroxybibenzyl.
The Sub‐Cellular Localisation of Pyruvate Carboxylase and of Some Other Enzymes in Aspergillus nidulans
Authors: OSMANI Stephen A. , SCRUTTON Michael C. 
Published: March 2005

The sub‐cellular localisation of enzymes has been defined by latency analysis, and fractionation by differential centrifugation, in cell‐free extracts prepared from the mycelium of Aspergillus nidulans by growth in the presence of 2‐deoxyglucose followed by treatment with a mixture of β‐glucuronidase, sulphatase and β‐glucanase and exposure to N2 cavitation at 5.2 PMa. In such extracts pyruvate carboxylase and NAD‐dependent and NADP‐dependent glutamate dehydrogenases are exclusively localised in the cytosol whereas all the other enzymes studied have sub‐cellular localisation patterns similar to those described for mammalian liver. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for many of the enzymes, e.g. NAD–malate dehydrogenase, NADP–isocitrate dehydrogenase, glutamate/oxaloacetate transaminase, fumarase, which show a marked extent of incomplete latency and the presence of significant activity in the mitochondrial and cytosolic fractions prepared by differential centrifugation. A novel method is described for detection of citrate synthase activity following electrophoresis of the cell‐free extract. Application of this method confirms the absence of a unique cytosolic isoenzyme of citrate synthase and hence shows that citrate synthase activity detected in the soluble fraction results from damage to the mitochondria during isolation.A scheme is proposed on the basis of these data to describe the organisation of lipid and amino acid synthesis from glucose in an organism which posessesses a cytosolic pyruvate carboxylase.
DNA Fragment Conformations
Authors: TRAN‐DINH Son , NEUMANN Jean‐Michel , TABOURY Jean , HUYNH‐DINH Tam , RENOUS Sylvie , GENISSEL Bernadette , IGOLEN Jean 
Published: March 2005

The conformation and the helix‐coil transition of two complementary hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), were investigated as a function of temperature by 1H‐NMR spectroscopy at 500 MHz and 90 MHz. At high temperature (t > 70–80°C), in the coil form, the proton chemical shifts depend strongly on the nature and position in the sequence of the residue. Purine and pyrimidine residues can be distinguished not only by the difference in chemical shift of the H 2′ and H 2” resonances but also by that of the H 3′ or H 4′ protons. For equivalent positions, the dA proton resonances are always located at lower fields than those of other residues. For residues of the same nature the chemical shift of the H 3′ or H 4′ protons decreases in the following order: δ(pdNp) > δ(dNp) > δ(pdN), where dN = dA, dG, dC or dT; moreover the terminal residue can easily be identified by either the H 1′ resonance [δ(pdN) > δ(pdNp, dNp)] or the H 2” resonance for the purine family [δ(pdN) < δ(pdNp, dNp) ≤δ(pdNp, dNp, pdN), where dN = dA, dG] and the H 2′ resonance for the pyrimidine family [δ(pdN) > δ(pdNp, dNp), where dN = dC or dT].The chemical shift difference between the coil and helix forms is generally greater for the pyrimidine than for the purine residues and the proton resonances of the external residues often exhibit different temperature‐dependent profiles. The results suggest that, as in the helical form the bases of adjacent residues are close to each other, the pyrimidine base proton resonances are shifted not only by the change in environment but also by the ring‐current effects from neighbouring purine bases. In the intercalated purine‐pyrimidine sequences, the protons of an internal pyrimidine residue are statistically subjected to opposite ring‐current effects from the base of the preceding and following residues. The difference in temperature‐dependent behaviour of the H 2” resonances between the initial or internal purine residues (where the phosphate group is located at the 3′ position) on the one hand and the terminal residue on the other can be explained by the change in conformation of the phosphate groups from the coil to the helix form.From measurements of proton coupling constants and observation of the NH resonances, it was concluded that the duplexes of both hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), adopt the B‐helical conformation and open sequentially from the exterior with increasing temperature. The midpoint temperatures, t1/2, coil proportions and helix‐coil dissociation constants Kd, were determined from the melting curves. The dissociation rate constant, kd, was deduced versus temperature from the line width of the H 1′ and H 5 resonances. The midpoint temperature of each residue and the difference between the t1/2 external and central residues depend strongly on the number of the dG · dC base pairs and their positions in the sequence, whereas the dissociation enthalpy appears to be independent for sequences of the same size.
Synthesis of Rat Liver Lactate Dehydrogenase and Characterization of Its mRNA
Authors: EISENMANN Andreas , SCHMELZER Elmon , NORTHEMANN Wolfgang , PHILLIPS John , KAISER Clemens , WITT Irene , DECKER Karl , HEINRICH Peter C. 
Published: March 2005

Rat liver lactate dehydrogenase (LDH) has been synthesized in hepatocytes and in a cell‐free translation system. The subunit synthesized in both systems displayed the same electrophoretic mobility upon sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. Sequence analysis of the subunits translated in vitro and synthesized in vivo indicated N‐acetyl‐alanyl‐alanine for both N termini. Thus, the newly synthesized subunit does not exhibit an amino‐terminal extension.The mRNA for the lactate dehydrogenase subunit was exclusively found in free polysomes. A size of 2120 ± 240 nucleotides was estimated for the mRNA. Only about 50% of these nucleotides are needed to code for the polypeptide chain of the enzyme.
The Simian‐Virus‐40 Large‐Tumor Antigen in Replicating Viral Chromatin
Authors: STAHL Hans , BAUER Michael , KNIPPERS Rolf 
Published: March 2005

The large tumor antigen (T antigen) is a genome regulation protein, coded by simian virus 40, that binds with high affinity to specific binding sites on viral DNA. The specifically bound T antigen is released from these sites in 0.2–0.3 M NaCl. Immunoprecipitation techniques were used to show that T antigen also dissociates in 0.2–0.3 M NaCl from mature viral chromatin but not from replicating viral chromatin. In fact, a considerable fraction of T antigen remains associated with replicating chromatin at NaCl concentrations as high as 1.2 M NaCl when most chromatin proteins, including histones, dissociate. However, T antigen binding to both replicating DNA and mature DNA is sensitive to intercalating drugs such as caffeine and ethidium bromide.We consider the possibility that the unexpectedly tight binding of T antigen to replicating DNA is related to the function that T antigen performs during viral DNA replication.
Heme P460 of Hydroxylamine Oxidoreductase of Nitrosomonas
Authors: HOOPER Alan B. , DEBEY Pascale , ANDERSSON Kristoffer K. , BALNY Claude 
Published: March 2005

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO‐binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2‐oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate‐limiting. Activation of dioxygen for N‐oxidation by ferrous HAO may involve the two‐electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen.Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 × 10−3 M−1 s−1 and Kd, 12μM.
On the Transhydrogenase Activity of Baker's Yeast Flavocytochrome b2
Authors: URBAN Philippe , LEDERER Florence , ALLIEL Patrick M. 
Published: March 2005

It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction.It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control.Determinations of the steady‐state deuterium isotope effect show that the rate‐limiting step in the oxidation of halogenolactates is abstraction of the α‐hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate‐limiting step in the reverse reaction must be protonation of the putative carbanion.
Somatic Antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander A. , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , STANISLAVSKY Evgeny S. , MASHILOVA Galina M. 
Published: March 2005

On mild acid degradation of Pseudomonas aeruginosa O:3(a),c, O:3a,d,e and O:3(a),d,f lipopolysaccharides O‐specific polysaccharides were isolated. The trisaccharide repeating units of O:3(a),c and O:3a,d,e polysaccharides contained 2‐acetamido‐2,6‐dideoxy‐d‐galactose and 2,3‐(1‐acetyl‐2‐methyl‐2‐imidazolino‐5,4)‐2,3‐dideoxy‐d‐mannuronic acid, which were identified previously as the constituents of P. aeruginosa O:3a,b and O:3a,d O‐specific polysaccharides, as well as 2,3‐diacetamido‐2,3‐dideoxy‐l‐guluronic acid, which has never before been found in nature. The last monosaccharide was identified without being isolated in the free state, by means of 13C nuclear magnetic resonance spectroscopy.The structures of O:3(a),c and O:3a,d,e polysaccharides were established by selective hydrogen fluoride solvolysis followed by modification of the trisaccharides obtained and analysis of the 13C nuclear magnetic resonance spectra at each modification stage. The polysaccharides possessed similar structures of repeating units differing from each other in the anomeric configuration of the N‐acetylfucosamine residue only:→4)DManImU(β1→4)LGul(NAc)2U(α1→3)DFucNAc(β1‐ and →4)DManImU(β1→4)LGul(NAc)2U(α1→3)DFucNAc(α1‐,where DManImU = 2,3‐(1‐acetyl‐2‐methyl‐2‐imidazolino‐5,4)‐2,3‐dideoxy‐d‐mannuronic acid, LGul(NAc)2U = 2,3‐diacetamido‐2,3‐dideoxy‐l‐guluronic acid, DFucNAc = 2‐acetamido‐2,6‐dideoxy‐d‐galactose.The same components were detected in the O:3(a),d,f polysaccharide but not in the same stoichiometric ratio; this polysaccharide possessed no regular structure.The immunochemical study of lipopolysaccharides of all five P. aeruginosa O:3 serotypes showed a definite interrelation between their serological properties and structures of the corresponding O‐specific polysaccharides.
Immunochemical Properties of Vertebrate α‐Crystallins
Authors: PURI Nirdosh , AUGSTEYN Robert C. , OWEN Elisabeth A. , SIEZEN Roland J. 
Published: March 2005

A competitive radioimmunoassay was used to determine the reactivities of α‐crystallins from 13 species with antibodies directed toward calf α‐crystallin. The results indicate that species as diverse as human and dogfish share the same number of crossreacting antigenic determinants. The various α‐crystallins can be distinguished only on the basis of their differing affinities for the antiserum.Hydrophilicity profiles for αA and αB polypeptides of all species were found to be remarkably similar. On the basis of these, four major sequential determinants could be predicted for each polypeptide. The location and sequence of these determinants were found to be essentially conserved in all α‐crystallins examined. These results are in agreement with the observed crossreactivities. However, there was little obvious correlation between substitutions in determinants and observed variations in respective α‐crystallin/antibody affinities. Conservation of antigenic determinants over such a wide evolutionary range may reflect stringent constraints on the overall surface and three‐dimensional structure of vertebrate α‐crystallins.
Mitochondrial Carbamoyl Phosphate Synthetase Activity in the Absence of N‐Acetyl‐l‐glutamate
Authors: RUBIO Vicente , BRITTON Hubert Greenslade , GRISOLIA Santiago 
Published: March 2005

Rat liver carbamoyl‐phosphate synthetase I is shown to have synthetase and ATPase activity in the absence of acetylglutamate. Km values for ATP, Mg2+ and K+ are greatly increased, the Km for HCO−3 is not changed much, and the Km for NH+4 is markedly reduced. Vmax for the synthetase reaction is < 20% of that of the acetylglutamate‐activated enzyme whereas Vmax for the ATPase activity is > 40% of that with acetylglutamate. Pulse‐chase experiments with H14CO−3 show formation of less “active CO2” (the central intermediate) than with acetylglutamate; ATPase activity is reduced in proportion, but the synthetase activity is much smaller. Binding of one ATP molecule with high affinity (Kd=20–30μM) is shown in the absence of acetylglutamate. This appears to be the molecule of ATPB (ATPB provides the phosphoryl group of carbamoyl phosphate). In contrast, the affinity for ATPA (ATPA yields Pi) is much reduced. Initial velocity measurements without acetylglutamate show a time lag before reaching a constant velocity. At 50 μM acetylglutamate the lag is much longer, but at 10 μM acetylglutamate it is shorter. Activation by acetylglutamate requires ATP at concentrations sufficient to occupy the ATPA and the ATPB binding sites. Preincubation with 10 mM acetylglutamate alone shortens the activation time.From these findings we propose an allosteric model for activation of carbamoyl‐phosphate synthetase in which there are two active states, R and R · AcGlu. Binding of ATPA is associated with the conversion of T to R. R · AcGlu differs from R in that transfer to carbamate of the γ‐phosphoryl group of ATPB appears to be facilitated.
Characterization of the epidermal‐growth‐factor‐dependent phosphorylation system from normal mouse‐liver sinusoidal plasma membranes
Authors: EHRHART Jean‐Claude , ROLLET Emmanuelle , KOMANO Odile , CREUZET Claudine , LOEB Jacques 
Published: March 2005

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants.

The phosphorylation reaction was studied at 33°C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [γ‐32P]ATP or [γ‐32P]GTP essentially into one 170000 Mr protein. Some incorporation was observed in a minor 120000‐Mr component which appears to be a degradation product of the 170000‐Mr component. No EGF‐dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro.
The dephosphorylation of the 170000‐Mr component was observed after 4 min of incubation at 33°C. This dephosphorylation reaction was inhibited by addition of 5 mM p‐nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate.
The 170000‐Mr protein specifically bound 125I‐labeled EGF and thus appeared to be the hepatic EGF receptor.
The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170000‐Mr EGF receptor at 33°C. Tryptic peptide maps of the 32P‐labeled 170000‐Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P‐labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170000‐Mr protein after it was covalently linked to 125I‐labeled EGF showed only one 125I‐labeled peptide, the migration of which appeared different from that of 32P‐labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170000‐Mr protein, labeled with 32P or 125I‐EGF.
Solubilization and characterization of an angiotensin II binding protein from liver
Authors: SEN Indira , JIM Kam F. , SOFFER Richard L. 
Published: March 2005

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II.Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high‐affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high‐affinity binding sites (Kd= 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure‐activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non‐physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68000 by polyacrylamide gel electrophoresis.