bluuebunny/crossref_metadata_2025_split huggingface dataset:
The asparaginyl‐tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316
Authors: AOKI Hiroyuki , YAWORSKY Paul J. , PATEL Samir D. , MARGOLIN‐BRZEZINSKI Denise , PARK Kyung S. , GANOZA M. Clelia 
Published: March 2005

Escherichia coli strain N4316 is a mutant that exhibits temperature‐sensitive growth at 43°C and temperature‐sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called ‘rescue’, isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild‐type parental strain; the purified protein is a homodimer of 54 kDa.The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature‐sensitive growth phenotype of the mutant at the non‐permissive temperatures. The cloned gene was sequenced, mapped to the 20.9‐min region of the E. coli chromosome and shown to code for a 466‐amino‐acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl‐tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read‐through of nonsense codons and protein copy number is discussed.
Ovohemerythrin, a major 14‐kDa yolk protein distinct from vitellogenin in leech
Authors: BAERT Jean‐Luc , BRITEL Mostafa , SAUTIERE Pierre , MALECHA Jean 
Published: March 2005

A 14‐kDa protein was identified as a major component of mature oocytes of the leech Theromyzon tessulatum. This protein was, like vitellin, detected in the content of yolk granules and was purified by gel‐permeation and ion‐exchange chromatography. The yolk protein corresponded to an iron‐binding protein which exists in a monomeric unglycosylated form and had no similarities to vitellin. However, a strong resemblance between this protein and sipunculid hemerythrin, a non‐heme iron‐binding protein, was observed on the basis of its characteristics including molecular mass, iron content, ultraviolet/visible spectrum, amino acid composition and N‐terminal sequence. These similarities with hemerythrin and the accumulation of the protein in the oocyte justify the name ovohemerythrin given to the molecule.A coelomic‐fluid protein immunologically related to ovohemerythrin was detected in vitellogenic animals. The protein was purified with the chromatographic procedure used to isolate ovohemerythrin from oocytes and was found to be similar to the oocyte protein. This circulating ovohemerythrin was present in large amounts in the coelomic fluid while gametogenesis is in progress, i. e. after the third and last blood meal of the animal (stage 3), except at the time of oocyte enlargement when its concentration decreases dramatically. However, in contrast to vitellogenin, which is detected specifically in the coelomic fluid of leeches at stage 3, circulating ovohemerythrin is also observed after the first (stage 1) and second (stage 2) blood meal. This observation suggests a more complex function for ovohemerythrin than being merely a yolk nutrient for the embryo.
Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane‐bound malic enzyme
Authors: KENDRICK Andrew , RATLEDGE Colin 
Published: March 2005

The component fatty acids of the endogenous phospholipids of microsomal preparations of Mucor, when shaken at 30°C, increased in both chain length and in degree of unsaturation. The net effect was the production of γ‐linolenic acid which, over 2 h, increased from 17% to 32% of total fatty acids present. No further significant changes occurred after this time.
The major site for desaturation/elongation reactions was at the sn‐2 position of Ptdlns. PtdCho and PtdEtn were not implicated.
Of numerous metabolites and cofactors added to the microsomes, only malate could prolong the elongation/desaturation reactions for up to 6 h. This effect was shown to be due to a membrane‐associated malic enzyme [malate dehydrogenase (decarboxylating) NADP+] with the NADPH produced being used in fatty‐acid desaturation.
Kinetic analysis of cytosolic and microsomal enzymes [both in 0.1% (mass/vol.) Chaps] could not distinguish between them. However, when the microsomal malic enzyme was dialysed to remove Chaps, it lost 90% of activity, although the cytosolic malic enzyme lost only 20% activity.
The structural analogue of malate, tartronic acid, which is an inhibitor of malic enzyme, also inhibited the malate‐induced stimulation of fatty‐acyl group desaturation and elongation in the microsomal membranes.
It is concluded that two distinct malic enzymes exist, one soluble and one membrane bound, with similar active sites. Both have different roles in the production of NADPH, for lipid metabolism. The former will produce NADPH for fatty‐acid biosynthesis whilst the latter produces NADPH for fatty‐acid desaturation.
β‐Adrenergic stimulation of C6 glioma cells: effects of cAMP overproduction on cellular metabolites
Authors: PIANET Isabelle , CANIONI Paul , LABOUESSE Julie , MERLE Michel 
Published: March 2005

We used 31P‐NMR spectroscopy to investigate the response of living C6 glioma cells to stimulation by a β‐adrenergic agonist, isoproterenol. In the presence of 3‐isobutyl‐1‐methylxanthine, stimulation induced an accumulation of cAMP, making possible the NMR detection of the second messenger in living cells grown on microcarrier beads and perfused in the NMR tube. The cAMP signal rose to a maximum level within 20–25 min of stimulation; thereafter it decreased to the detection threshold within 60 min. At the same time, 40% increases of phosphomonoester and diphosphodiester signals were observed, whereas no significant change in phosphocreatine and nucleotide signals was detected. The kinetics of changes of the cellular content in phosphorylated metabolites were analyzed after recording 31P‐NMR spectra of cell perchloric acid extracts as a function of time of stimulation. cAMP accumulation in stimulated cells was evidenced by a near linear increase of its NMR signal as a function of incubation time (from 0 to 60 min). Concomitantly with the production of cAMP, the data showed 30% decreases of phosphocreatine and ATP levels within 60 min of stimulation, and an unexpected redistribution of pyrimidine and purine nucleoside triphosphates. At the same time, levels of phosphomonoesters (phosphorylcholine and phosphorylethanolamine) and phosphodiesters (glycerophosphorylcholine and glycerophosphorylethanolamine) rose (50% increase). 13C‐NMR spectra of cell perchloric acid extracts prepared after isoproterenol stimulation of cells incubated in the presence of [1‐13C]glucose indicated a higher glucose content in stimulated cells, whereas the resonance of ribose C1 was diminished. Moreover, the resonances of C1 of ethanolamine and choline (and their derivatives) were increased in spectra of stimulated cells, whereas that of C3 of serine was decreased. In addition, the 13C‐NMR data indicated that neither the pattern of glutamate carbon enrichment nor the glutamate/glutamine ratio was modified in stimulated cells. On the other hand, the heteronuclear coupling pattern of the lactate (methyl group) resonance in 1H‐NMR spectra of cell incubation media indicated that no change occurred in the carbon flux through the pentosephosphate shunt under stimulation. The results of this multinuclear NMR approach are discussed in terms of metabolic responses of C6 cells to β‐adrenergic stimulation and cAMP overproduction.
Molecular basis of low‐temperature sensitivity in pig hemoglobins
Authors: CONDÒ Saverio G. , CORDA Marcella , SANNA Maria T. , PELLEGRINI Maria G. , RUIZ Maite Prats , CASTAGNOLA Massimo , GIARDINA Bruno 
Published: March 2005

It has been generally assumed that mammals have blood with a greater temperature sensitivity than ectothermic organisms. Recent results have shown that in some species of mammals, Hb displays a value of overall oxygenation enthalpy (δH) much less exothermic than that observed for most mammalian hemoglobins, including human adult Hb. In this respect, a very interesting case is represented by porcine blood which shows a modest effect of temperature, the temperature coefficient of its oxygen‐dissociation curve being significantly lower than that of human blood.Here we report a detailed functional characterization of pig Hb, which, interpreted on the basis of the amino acid sequence of the α and β chains of the molecule, sheds some light on the molecular basis of the phenomenon.
Stopped‐flow fluorescence and steady‐state kinetic studies of ligand‐binding reactions of glucoamylase from Aspergillus niger
Authors: OLSEN Karsten , SVENSSON Birte , CHRISTENSEN Ulla 
Published: March 2005

The presteady‐state and steady‐state kinetics of the binding and hydrolysis of substrates, maltose and isomaltose, and the transition‐state analogue, gluconolactone, by glucoamylase from Aspergillus niger were investigated using initial‐rate, stopped‐flow and steady‐state methods. The change in the intrinsic fluorescence of the enzyme was monitored. Distinct mechanistic differences were observed in the interaction of the enzyme with maltose compared to isomaltose. Hydrolysis of maltose requires a three‐step mechanism, whereas that of isomaltose involves at least one additional step. The rates of an observed conformational change, which is the second discernible step of the reactions, clearly show a tighter binding of maltose compared to isomaltose, probably because the reverse rate constants differ. Compared to the non‐enzymic hydrolysis the transition‐state stabilization energy of glucoamylase is approximately −66 kJ/mol with maltose and only −14 kJ/mol with isomaltose.Kinetic analysis of the binding of the inhibitor, gluconolactone, implies that independent interactions of two molecules occur. One of these, apparently, is a simple, fast association reaction in which gluconolactone is weakly bound. The other resembles binding of maltose, involving a fast association followed by a conformational change. Based on the results obtained, we propose new reaction mechanisms for Aspergillus glucoamylase.
A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei
Authors: SCHMITZ Ruth A. , ALBRACHT Simon P. J. , THAUER Rudolf K. 
Published: March 2005

We have recently reported that the thermophilic archaeon Methanobacterium wolfei contains two formylmethanofuran dehydrogenases, I and II. Formylmethanofuran dehydrogenase II, which is preferentially expressed in tungsten‐grown cells, has been purified and shown to be a tungsten‐iron‐sulfur protein. We have now purified and characterized formylmethanofuran dehydrogenase I from molybdenum grown cells and shown that it is a molybdenum‐iron‐sulfur protein. The purified enzyme, with a specific activity of 27 U/mg protein, was found to be composed of three subunits of apparent molecular mass 64 kDa, 51 kDa, and 31 kDa and to contain per mol 146‐kDa molecule approximately 0.23 mol molybdenum, 0.46 mol molybdopterin guanine dinucleotide, and 6.6 mol non‐heme iron but no tungsten (<0.01 mol). The molybdenum enzyme differed from the tungsten enzyme (8 U/mg) in that it catalyzed the oxidation of N‐furfurylformamide and formate and was inactivated by cyanide. The two enzymes also differed significantly in the pH optimum, in the apparent Km for the electron acceptor, and in the chromatographic behaviour. The molybdenum enzyme and the tungsten enzyme were similar, however, in that the N‐terminal amino acid sequences determined for the α and β subunits were identical up to residue 23, indicating that the two proteins are isoenzymes.The molybdenum enzyme, as isolated, was found to display an EPR signal derived from molybdenum as evidenced by isotope substitution.
Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca2+ ‐ATPase
Authors: de FORESTA Béatrice , HENAO Fernando , CHAMPEIL Philippe 
Published: March 2005

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca2+‐ATPase and the non‐ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 °C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 × 105 (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids.Non‐solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65–70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium‐saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate‐limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding‐induced transition of nonphosphorylated ATPase.Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5–5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired. These results establish dodecylmaltoside as a potentially useful tool for studying the interaction between the two Ca2+ binding sites of membranous SR ATPase and their coupling with the catalytic site of ATPase.
The use of multiple‐pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti‐(recombinant bovine growth hormone) sera
Authors: BEATTIE James , FAWCETT Hilary A. C. , FLINT David J. 
Published: March 2005

A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth‐hormone (rbGH) molecule recognised by three polyclonal guinea‐pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea‐pig antisera, 3 or 4, very‐well‐defined major continous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea‐pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring α‐helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea‐pig antiserum.Comparison of the four guinea‐pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson‐Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods.In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous epitopes defined by different anti‐rbGH sera, there are large regions of overlap defining immunogenic core regions within the molecule. We believe that studies of this nature, together with further understanding of antigen processing and peptide presentation to immune cells, may have a role to play in the development of candidate peptide vaccines.
Nuclear‐magnetic‐resonance analysis of the capsular antigen of Actinobacillus pleuropneumoniae serotype 9
Authors: BEYNON Linda M. , RICHARDS James C. , PERRY Malcolm B. 
Published: March 2005

The specific capsular antigen of Actinobacillus pleuropneumoniae serotype 9 was characterized by one‐dimensional and two‐dimensional high‐field nuclear‐magnetic‐resonance methods, and by chemical analyses, as a teichoic‐acid‐type polymer of a repeating unit having the structure

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The basic polymer structure is identical to capsular antigens of Neisseria meningitidis group H, Escherichia coli K62 (K2ab) and Pasteurella haemolytica serotype T15.
Effect of 2‐mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin‐secreting cells
Authors: JANJIC Danilo , WOLLHEIM Claes B. 
Published: March 2005

The role of glutathione (GSH) in the differentiated state of insulin‐secreting cells was studied using 2‐mercaptoethanol as a means of varying intracellular GSH levels. 2‐Mercaptoethanol (50 μM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS‐1, the latter being dependent on the presence of 2‐mercaptoethanol for survival in tissue culture. The effect of 2‐mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2‐mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine‐uptake in INS‐1 cells. At equimolar concentrations to cystine, 2‐mercaptoethanol caused stimulation of [35S]cystine‐uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc− carrier system. INS‐1 cells with a high GSH level, cultured 48 h with 2‐mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content.The effect of variations of the GSH levels on short‐term insulin release was studied. No alteration of glyceraldehyde‐induced or KCl‐induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS‐1 cells, a high GSH level was associated with a slighly lower insulin release. In INS‐1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS‐1 cells no change in the GSH/GSSG ratio was detected during glucose‐induced or 3‐isobutyl‐1‐methylxanthine‐induced insulin release.In conclusion, 2‐mercaptoethanol‐dependent INS‐1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2‐mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin‐secreting cells.
Mechanism of carboxypeptidase‐Y‐catalysed peptide semisynthesis
Authors: CHRISTENSEN Ulla , DRØHSE Helle B. , MØLGAARD Lone 
Published: March 2005

The initial rate steady‐state kinetics of carboxypeptidase‐Y‐catalyzed hydrolysis and aminolysis reactions with some α‐N‐benzoyl‐l‐tyrosinyl compounds has been investigated using L‐valinamide as the nucleophile in aminolysis. Hydrolysis of α‐N‐benzoyl‐l‐tyrosinyl ethyl ester, 4‐nitroanilide, and‐amide has been studied in the pH range 4–9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme‐substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate‐determining with the ester substrate, but with the 4‐nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate‐determining step is apparent in the range pH 4–9. For the 4‐nitroanilide and the amide substrates, the kinetic paramcter, kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active‐site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate.Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L‐valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L‐valinamide on the initial rate behaviour of carboxypeptidase‐Y‐catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl‐enzyme complex forming an enzyme‐aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined.The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrates. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.