bluuebunny/crossref_metadata_2025_split huggingface dataset:
Méthionyl‐tRNA synthétase des embryons de blé: dissociation en sous‐unités
Authors: CHAZAL Philippe , THOMES Jean‐Claude , JULIEN Raymond 
Published: March 2005

Methionyl‐tRNA Synthetase from Wheat Embryo: Dissociation into Subunits Wheat‐embryo methionyl‐tRNA synthetase is a dimeric protein of β2 structure. When highly diluted, it loses the capacity to catalyze ATP‐[32P]PPi exchange and to aminoacylate tRNAMet: at low enzymatic concentrations the rates of formation of [32P]ATP and [14C]methionyl‐tRNAMet are lower than those predicted by extrapolating the rates determined at higher enzyme concentrations. The difference between observed and expected rates becomes greater with decreasing enzyme concentration.Filtration of purified, dilute enzyme preparations on Sephadex G‐200 results in the separation of dimer and monomer fractions. The proportion of monomer present increases with increasing pre‐incubation times before the assay and demonstrates an equilibrium between active dimers and inactive monomers. The above‐mentioned loss of enzymatic activity is explained by the equilibrium being shifted towards the production of monomers. Data previously gathered for Escherichia coli prolyl‐tRNA synthetase and for bovine‐pancreatic tryptophanyl‐tRNA synthetase, coupled with the present results, suggests that the dissociation of dimeric synthetases may be a general phenomenon in eukaryotes as well as in prokaryotes.The number of sub‐units and the dissociation constant were obtained at equilibrium, according to relations adapted to the case of oligomeric enzymes (KD≃ 13 nM at 25 °C and pH 7.5).Rate constants were determined by kinetic studies of the attainment of equilibrium. The rate constant k1 for monomolecular dissociation was determined to be 1.85 · 10−3 s−1 and k2 for the bimolecular association to be 0.145 · 106 M−1 s−1. The KD calculated from k1 and k2 was coherent with the experimentally determined value, at equilibrium.The sub‐unit interactions, which involve only a small quantity of energy (ΔG°≃+ 11 kcal mol−1; +45 kJ mol−1) at 25 °C, depend on the ionic environment of the medium and the presence of substrates. Alkaline pH favors monomer production, while the presence of methionine, (Mg‐ATP)2− and tRNAMet protect the synthetase from dissociation. 2‐mercaptoethanol and dithioerythritol prevent only slightly the loss of activity. Bovine serum albumin, however, protects the enzyme from dissociation under dilute conditions.
Prostaglandin Production by Normal and Transformed 3T3 Fibroblasts in Cell Culture
Authors: HAMMARSTRÖM Sven 
Published: March 2005

Prostaglandin E2 and prostaglandin F2α were quantitatively determined in culture media from Balb/c 3T3 fibroblasts and two virus‐transformed derivatives of these cells. Regular and (simian virus 40)‐transformed 3T3 cells produced low and almost identical prostaglandin E2 concentrations. The levels were maximal (10 ng/ml) 24 h after planting and decreased during the next 96 h to 2.5 ng/ml. In contrast, polyoma‐virus‐transformed 3T3 cells produced prostaglandin E2 continuously for 120 h, giving a final concentration of 270 ng/ml.Fresh medium supplemented with calf serum reinitiated prostaglandin production in confluent 3T3 cells. Maximal prostaglandin E2 concentrations were obtained between 6 and 18 h after medium change and were proportional to the serum concentration of the medium. Indomethacin (10−6 M) completely inhibited prostaglandin E2 production but not prostaglandin F2α production by these cells.
RNA Methylation and Control of Eukaryotic RNA Biosynthesis
Authors: CABOCHE Michel , BACHELLERIE Jean‐Pierre 
Published: March 2005

The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations.No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45‐S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed.At the post‐transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45‐S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected.Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long‐term labelling studies. (b) The rate of appearance of ribosomal 28‐S RNA into the cytoplasm is severely affected (85–90%) inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28‐S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm).Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.
The Interaction of Phospholipid Membranes and Detergents with Glutamate Dehydrogenase
Authors: NEMAT‐GORGANI Mohsen , DODD George 
Published: March 2005

The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg‐yolk, and with beef brain phosphatidylserine was investigated by steady‐state kinetic methods.
The phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min.
The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non‐competitive interaction with Ki(app) values varying between (0.9–6.1) × 10−6 M.
The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme · lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid.
Sodium dodecylsulphate causes a time‐dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied.
The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.
The Interaction of Phospholipid Membranes and Detergents with Glutamate Dehydrogenase
Authors: NEMAT‐GORGANI Mohsen , DODD George 
Published: March 2005

Both the anionic detergent sodium dodecylsulphate and the cationic detergent cetyltrimethylammonium bromide quenched the protein fluorescence of glutamate dehydrogenase. The anionic compound was more effective and brought about 50% quenching at a detergent concentration of 0.4 mM. The zwitterionic amphiphile, lysolecithin, did not quench the protein fluorescence and neither did the short‐chain detergent n‐hexylsulphonate, which under the range of concentrations examined (< 1 mM) does not form micelles.
The zwitterionic phospholipid, phosphatidylcholine, did not quench the protein fluorescence but the anionic phospholipids, phosphatidylserine and cardiolipin, induced a reversible quenching of the enzyme fluorescence. These observations confirm the specificity of the phospholipid–enzyme interactions as deduced from the kinetic studies of the preceding paper. The degree of quenching brought about by the phospholipids decreased with increasing ionic strength and increasing pH and could be substantially reduced by basic proteins. An electrostatic contribution to the interaction is inferred from these results.
The binding of the anionic phospholipids to the enzyme is manifested in a further enhancement of the fluorescence of a 1‐anilinonaphthalene‐8‐sulphonate · enzyme complex. The presence of substrates and allosteric effectors affect the interaction of the lipids with the enzyme as indicated by the magnitude of this increase in fluorescence. The enhancement of fluorescence of NADH when bound to the enzyme was not affected by the binding of the lipids.
The complex formed between the enzyme and phosphatidylserine/phosphatidylcholine can be solubilized in isooctane. The photolability of the aqueous protein when subjected to irradiation at 280 nm is suppressed in the isooctane‐soluble complex.
Phosphatidylserine brings about a rapid (t1/2 is about 150 ms at a lipid concentration of 0.75 mM) dissociation of the linear aggregates formed between the enzyme oligomers.
A model of the enzyme · lipid‐membrane complex, consistent with these results, is proposed. It is suggested that the enzyme is an allotopic protein and that the dissociation of the enzyme in vitro may involve binding sites on the protein which are designed for interaction with the cardiolipin of the inner mitochondrial membrane, when the enzyme is in the mitochondrial matrix.
UDP‐Apiose/UDP‐Xylose Synthase
Authors: MATERN Ulrich , GRISEBACH Hans 
Published: March 2005

The UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley has been purified 1400‐fold by an improved method. The ratio of apiose to xylose formed from UDP‐D‐glucuronic acid (UDP‐GlcUA) remained constant throughout the purification procedure. Dodecylsulfate‐gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE‐cellulose at different pH‐values, and on ω‐aminoalkyl‐Sepharose, and by gel filtration on Acrylex P‐100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N‐terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids.Separation of the two proteins was achieved on DEAE‐cellulose in the presence of urea. After dialysis only the 86000‐Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000‐Mr protein enzyme stability was decreased drastically.By equilibrium dialysis it was found that 0.5 mol UDP‐GlcUA are bound per mole of 86000‐Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4‐keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C‐3 of the 4‐ulose intermediate prior to ring contraction to apiose was found.
Transport of Hexoses across the Liver‐Cell Membrane
Authors: BAUR Hartmut , HELDT Hans W. 
Published: March 2005

The uptake of radioactively labelled hexoses into the cellular space of isolated liver cells has been studied using silicone layer filtering centrifugation. From the hexoses tested, D‐glucose is transported most rapidly followed by D‐galactose and D‐fructose. The rate of L‐glucose transport is only 5% of that of D‐glucose. This transport enables the concentration of free aldohexoses in the cellular space to reach the external concentration.For D‐glucose the rate of transport into the cell largely exceeds the rate of metabolic conversion. This is different with D‐fructose where the rate of transport is considerably lower but the rate of metabolism higher. Here the transport may even be a limiting step.The transport of D‐glucose and D‐galactose shows a saturation characteristic, whereas D‐fructose appears not to be saturatable. The Km for D‐glucose is found to be 30 mM. All these hexoses compete with each other for transportation.The temperature dependency of D‐glucose reveals an activation energy of 22 kcal (92 kJ)/mol (4–18 °C) and 7 kcal (29 kJ)/mol (18–37 °C). The transport of D‐glucose, D‐galactose and D‐fructose in inhibited by cytochalasin B, phloretin and phlorizin. It is not dependent on Na+ ions. Preliminary results showed no stimulation of the transport by insulin.It is concluded that D‐glucose and other hexoses are transported by carrier‐mediated diffusion across the plasma membrane of liver cells. This transport shows a large resemblance to the transport of D‐glucose into human erythrocytes.
4‐Amino‐hex‐5‐enoic Acid, a Selective Catalytic Inhibitor of 4‐Aminobutyric‐Acid Aminotransferase in Mammalian Brain
Authors: LIPPERT Bruce , METCALF Brian W. , JUNG Michel J. , CASARA Patrick 
Published: March 2005

Incubation of rat brain 4‐aminobutyrate aminotransferase with 4‐amino‐hex‐5‐enoic acid, a substrate analog of 4‐aminobutyric acid, results in a time‐dependent irreversible loss of enzymatic activity. In the presence of 0.1 mM inhibitor the half‐life of the inactivation process is approximately 6 min. Low concentrations of L‐glutamic acid or 4‐aminobutyric acid protect against this inactivation. while 2‐oxoglutarate prevents this protection, suggesting that only the pyridoxal form of the enzyme is susceptible to inhibition by 4‐amino‐hex‐5‐enoic acid.The irreversible inhibition of mammalian 4‐aminobutyrate aminotransferase by 4‐amino‐hex‐5‐enoic acid is selective. There is no inhibition of this enzyme from Pseudomonas fluorescens with the inhibitor at mM concentrations. Even at 10 mM there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited over 500 times more slowly than rat brain 4‐aminobutyrate transaminase.
Properties of ATPase Activity in Coupling Factor from Chromatium Strain D Chromatophores
Authors: GEPSHTEIN Amira , CARMELI Chanoch 
Published: March 2005

Coupling factor extracted from chromatophores of the photosynthetic bacteria Chromatium strain D was partially purified. The enzyme catalyzed ATPase activity in the presence of Ca2+ and Mg2+ ions. Higher Vapp values were obtained when the activity was measured as a function of the divalent cation‐ATP complex rather than as a function of either the divalent cation or ATP because the free components competitively inhibited the activity in the presence of the cation‐ATP complex. The Km values were lower than or equal to the Ki values for free ATP indicating that the cation‐ATP complex is bound tighter than the free ATP to the enzyme. Based on these results a possible mode of binding of substrate to the active site of the enzyme was suggested.A comparative study indicated no changes in the temperature dependance of ATPase activity when the enzyme was solubilized. However, possible conformation changes could have caused a decrease in the Km values for the (Ca‐ATP)2− and (Mg‐ATP)2− and in the Ki for free Mg2+ ions and ATP. The Ki for free Ca2+ ions increased on solubilization of the coupling factor. ATPase activity was inhibited by dicyclohexylcarbodiimide both in the soluble and in the membrane‐bound coupling factor.
Biosynthesis of Cyclopentenyl Fatty Acids
Authors: CRAMER Udo , SPENER Friedrich 
Published: March 2005

In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non‐proteinogenic amino acid may serve as precursor of cyclo‐pentenyl fatty acids via aleprolic acid, the starter molecule for these long‐chain compounds.After administration of cyclopentenyl[2‐14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1‐14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1‐14C]acetate revealed that the chain‐lengthening systems for straight‐chain and cyclic fatty acids were still functioning in mature seeds.Endosperm and embryo of H. anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2‐14C]glycine, [1‐14C]aleprolic acid and [1‐14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine → aleprolic acid → cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2‐units to cyclopentenyl fatty acids.
Measurement of Progesterone and Pregnenolone 16α‐Hydroxylase Activities by a Tritium‐Exchange Method
Authors: DE GRAEVE Jean , KREMERS Pierre , GIELEN Jaques E. 
Published: March 2005

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group, has been developed for the measurement of progesterone and pregnenolone‐16α‐hydroxylase activity. The incubation medium consists of a phosphate buffer (pH 7; 150 mM), NADPH (1 mM), nicotinamide (10 mM) and magnesium chloride (4 mM). Tween‐80 (1 mg/ml) is used to solubilize saturating concentrations of [16‐3H]progesterone (200 μM) or [16‐3H]‐pregnenolone (50 μM). The microsomal fraction isolated from a male rat liver is used as the enzymic source.The enzymically released tritium is recovered in the incubation medium as molecules of tritiated water which are distilled under reduced pressure. The amount of radioactivity present in the water exactly reflects the 16α‐hydroxylase activity.The method is easy to perform and is completely independent of any further metabolism of the 16α‐hydroxylated products.
Role of Divalent Ions in Folding of tRNA
Authors: LEROY Jean‐Louis , GUÉRON Maurice , THOMAS Gilles , FAVRE Alain 
Published: March 2005

The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4‐thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the ‘Y’ base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8–13 photoproduct (in which 4‐thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed.In low salt (e.g. 4.5 mM Na+), the region of 4‐thiouracil is affected strongly as the first eight Mn2− bind to tRNA, whereas the fluorescence of the ‘Y’ base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration.Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese‐phosphate binding supplemented by electrostatic interaction with distant phosphates.
Regulation of ATP Pools, rRNA and DNA Synthesis in 3T3 Cells in Response to Serum or Hypoxanthine
Authors: GRUMMT Friedrich , PAUL Dieter , GRUMMT Ingrid 
Published: March 2005

The serum‐induced transition of 3T3 fibroblasts from resting to growing state is characterized by a marked increase in cellular ATP content, the maximal level of which is reached at the onset of DNA replication. This increase in cellular ATP during the G1 period of the cell cycle is correlated with about 3‐fold stimulation of transcription of rRNA measured in permeabilized cells in vitro. Addition of hypoxanthine to serum‐depleted quiescent 3T3 cells gives rise to an increase in both the ATP pool and the rate of rRNA synthesis. The expansion of cellular ATP pools after growth induction by serum seems to be a prerequisite for initiation of DNA synthesis since inhibition of purine de novo biosynthesis by azaserine inhibits both ATP pool expansion and DNA replication. This effect of azaserine can be abolished by addition of hypoxanthine to the culture medium.It is concluded that (a) the increase of the rate of rRNA synthesis in 3T3 cells in response to growth factors or serum is controlled by the cellular purine nucleoside triphosphate concentration and (b) an increased ATP level is necessary for initiation of DNA synthesis but is not sufficient to trigger the events that lead to DNA replication.
Particle‐Bound Enzymes in the Bloodstream Form of Trypanosoma brucei
Authors: OPPERDOES Fred R. , BORST Piet , SPITS Hergen 
Published: March 2005

We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol‐3‐phosphate oxidase, ATPase, isocitrate dehydrogenase, acid phosphatase and part of the hyperoxide dismutase and malate dehydrogenase activities were found to be particle‐bound after fractionation of homogenates by differential centrifugation. Part of the ATPase activity was sensitive to oligomycin, an inhibitor of oxidative phosphorylation. This oligomycin‐sensitive activity can serve as a specific marker for the mitochondria.More than 80% of the NAD+ ‐linked glycerol‐3‐phosphate dehydrogenase in T. brucei was found to be particulate and latent. The enzyme could be activated by Triton X‐100, by the combined action of sonication and salt, but not by salt alone, and partially by freezing and thawing. We conclude that the NAD+ ‐linked glycerol‐3‐phosphate dehydrogenase is located inside an organelle.