bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies of Glutamate Dehydrogenase
Authors: RASCHED Ihab R. , BOHN Anneliese , SUND Horst 
Published: March 2005

Cross‐linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantely the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross‐linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8–9 of the 34 amino groups have reacted, only 2–3 of them are involved in cross‐links.Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross‐linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with monofunctional methylimidates, the loss of activity is reduced.Purified chymotryptic and tryptic peptides of the radioactive‐labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino‐terminal residue alanine‐1 and the others involve lysine‐105, lysine‐154, lysine‐269, lysine‐358 and lysine‐399.Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine‐105, lysine‐154, lysine‐269 and lysine‐358 participate in cross‐links, lysine‐269 and lysine‐358, respectively, being at isologous and lysine‐105 cross‐linked with lysine‐154 at heterologous contact domains of the enzyme.A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.
Poly(A)‐Containing RNA in Neuroblastoma: Immature and Differentiated Cells in Culture
Authors: CROIZAT Bernard , BERTHELOT Francis , FELSANI Armando , GROS François 
Published: March 2005

We have analysed the poly(A)‐containing RNA from neuroblastoma cells at two different developmental states: either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish.Suspension‐grown and monolayer cells were pulse‐labelled with tritiated uridine. The profile of cytoplasmic poly(A)‐containing RNA from suspension cells is highly heterogeneous with peaks ranging from 16–30 S. The profile obtained from differentiated cells appears somewhat distinct from the previous one. This is evidenced by a relative decrease in the 26‐S peak and a virtual disappearance of the 16‐S component.In order to compare the ‘steady‐state’ patterns of poly(A)‐containing RNA in these two developmental stages, polysomal RNA was prepared from unlabelled cells. Following sucrose gradient sedimentation, each fraction was hybridized to [3H]poly(U). Examination of the two RNA hybridization profiles reveals striking similarities suggesting that ‘steady‐state’ messenger populations include, on the average, the same subspecies. The 16‐S fraction, which was not observed after the pulse‐labelling of the monolayer culture, is detected here by hybridization to [3H]poly(U) when using polysomal poly(A)‐containing RNA from monolayer cells as substrate.These results suggest that terminal differentiation of neuroblastoma cells is not accompanied by major alterations of the transcription program and is paralleled by a marked stabilization of the 16‐S species.
Methionyl‐tRNA Synthetase from Escherichia coli: Substituting Magnesium by Manganese in the L‐Methionine Activating Reaction
Authors: HYAFIL François , BLANQUET Sylvain 
Published: March 2005

While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP‐PPi isotopic exchange reaction catalysed by methionyl‐tRNA synthetase from Escherichia coli, the latter ion was selected for a detailed analysis of the L‐methionine activation reaction. In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments. The thermodynamic parameters derived from steady‐state (ATP‐PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped‐flow) experiments are compared to those obtained in the presence of Mg2+ [Hyafil et al. (1976) Biochemistry, 15, 3678–3685].While the standard ΔG for the reaction (E · Met · ATP‐Me2+⇄ E · Met ∼ AMP · PPi−Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species. The ΔG for the formation of the E · Met ∼ AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex. Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi‐Me2+ from the E · Met ∼ AMP · PPi‐Me2+ species and from its abortive analog E · Met · Ado · PPi‐Me2+. Similarly the dissociation constant of ATP‐Me2+ from another dead‐end analog E · methioninol · ATP‐Me2+ is decreased by Mn2+. Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl‐tRNA synthetase is ruled out by the use of 7‐deazaadenosine.The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.
Cooperative and Antagonistic Interactions of Peptidyl‐tRNA and Antibiotics with Bacterial Ribosomes
Authors: CONTRERAS Antonio , VAZQUEZ David 
Published: March 2005

There is a single‐site interaction of [methylene‐14C]thiamphenicol and [methylene‐14C]chloramphenicol with run‐off ribosomes with dissociation constants Kd= 6.8 μM and Kd= 4.6 μM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A‐site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P‐site and others in the A‐site. The lower‐affinity bindings (dissociation constants Kd= 6.4 μM and Kd= 5.9 μM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A‐site. The higher‐affinity bindings (dissociation constants Kd= 2.3 μM and Kd= 1.5 μM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P‐site. Therefore binding of nascent peptides to the A‐site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P‐site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl‐tRNA is placed in the P‐site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics.There is a single‐site interaction per ribosome for [35S]thiostrepton, which does not appear to be affected by the attachment to the ribosomes of mRNA, tRNA and nascent peptides either to the A or the P‐site.[N‐methyl‐14C]Lincomycin, [N‐methyl‐14C]erythromycin, [G‐3H]streptogramin B and [G‐3H]‐streptogramin A bind to run‐off ribosomes and polysomes totally free from nascent peptides. However, these antibiotics do not interact with ribosomes bearing nascent peptides either in the A or the P‐site and therefore are not active on preformed polysomes. Thus lincomycin and streptogramin A only interact with free ribosomes and 50‐S subunits and block the early rounds of peptide bond formation prior to polysome formation. Erythromycin and streptogramin B do not inhibit either initiation or the first round of peptide bond formation. However, erythromycin and streptogramin B, prebound to the ribosome, block peptide elongation probably by steric hindrance with the growing oligopeptide chain when this reaches a certain critical length.
Subunit Structure of the Methionine‐Repressible Aspartokinase II–Homoserine Dehydrogenase II from Escherichia coli K12
Authors: DAUTRY‐VARSAT Alice , SIBILLI‐WEILL Lise , COHEN Georges N. 
Published: March 2005

The quaternary structure of Escherichia coli K 12 aspartokinase II–homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr= 176000. It is composed of two subunits having the same molecular weight and the same charge.The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine‐containing peptides and the uniqueness of the N‐terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II–homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I–homoserine dehydrogenase I.
Localization of Glycerol‐3‐Phosphate Oxidase in the Mitochondrion and Particulate NAD+‐Linked Glycerol‐3‐Phosphate Dehydrogenase in the Microbodies of the Bloodstream Form of Trypanosoma brucei
Authors: OPPERDOES Fred R. , BORST Piet , BAKKER Suzanne , LEENE Wolter 
Published: March 2005

We have studied the intracellular location of glycerol‐3‐phosphate oxidase and NAD+‐linked glycerol‐3‐phosphate dehydrogenase, two enzymes involved in the very active dihydroxyacetone phosphate:glycerol‐3‐phosphate cycle in Trypanosoma brucei. Isopycnic centrifugation of a large‐granule fraction resulted in a 10–20‐fold purification of the oxidase and showed that the enzyme behaved like the mitochondrial ATPase, isocitrate dehydrogenase and the particulate malate dehydrogenase. The activity profiles of these four enzymes in sucrose gradients were identical with maximal activity at a density of 1.17 g/cm3. Electron micrographs of a fraction equilibrating at this density only showed mitochondrial profiles.The particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase activity was found to band at a density of 1.23 g/cm3 in sucrose. A fraction equilibrating at this density was highly enriched in microbodies; it contained few mitochondrial profiles and less than 3% of the specific oxidase activity of the mitochondrial fraction. Because of the absence of microbody markers in T. brucei we turned to the insect trypanosome Crithidia luciliae, which contains a high activity of the microbody marker catalase. In C. luciliae part of the catalase activity bands at the same density as the particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase (Q = 1.26 g/cm3).From these results we conclude that in T. brucei bloodstream forms glycerol‐3‐phosphate oxidase is located in the mitochondrion and not in the microbodies, as proposed by a number of other investigators, and that the particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase is located within the microbodies. It is likely that this highly active dehydrogenase plays an essential role in glycolysis.
A Trypsin‐Resistant Fragment from Complexes of Ribosomal Protein S4 with 16‐S RNA of Escherichia coli and from the Uncomplexed Protein
Authors: NEWBERRY Veronica , YAGUCHI Makoto , GARRETT Roger A. 
Published: March 2005

A fragment of ribosomal protein S4 was prepared by limited trypsin digestion of a specific complex between protein S4 and 16‐S RNA. It was characterised for amino acid sequence and the N‐terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg‐43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA‐binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N‐terminal region of the protein is proposed and two probable functions of the excised region are given.Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg‐46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein · RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.
Diacylglycerol Acyltransferase from Rat Liver Microsomes
Authors: HOSAKA Kohei , SCHIELE Ulrich , NUMA Shosaku 
Published: March 2005

Diacylglycerol acyltransferase was resolved from rat liver microsomes and separated from glycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase. The separation was achieved by sucrose‐density‐gradient centrifugation of the enzyme preparation which was obtained by molecular‐sieve chromatography of microsomes solubilized with a nonionic detergent, Triton X‐100.
Although diacylglycerol acyltransferase and 1‐acylglycerolphosphorylcholine acyltransferase were not separated from each other, the two acyltransferases were distinguishable with respect to heat stability and sensitivity to sulfhydryl‐binding reagents.
Studies with the diacylglycerol acyltransferase preparation obtained have shown that this enzyme possesses a broad acyl‐donor specificity, utilizing saturated, monoenoic, dienoic and tetraenoic fatty acyl‐CoA thioesters efficiently.
A simplified assay method for diacylglycerol acyltransferase is described.
Investigation into the Occurrence and Structure of Lectin Receptors on Human and Bovine Erythrocyte, Milk‐Fat Globule and Lymphocyte Plasma‐Membrane Glycoproteins
Authors: NEWMAN Roland. A. , UHLENBRUCK Gerhard. G. 
Published: March 2005

The reduced disaccharide β‐d‐galactosyl(1‐3)‐N‐acetyl‐d‐galactosaminitol was released, after alkaline borohydride and neuraminidase treatment, from human and bovine erythrocyte membrane and fat globule membrane glycoproteins as well as from human and bovine lymphocyte plasma membranes. This disaccharide was found to be substituted to different extents by sialic acid in the various fractions. β‐d‐Galactosyl(1‐3)‐d‐acetyl‐d‐galactosamine is the immunodominant disaccharide of the Thomsen‐Friedenreich antigen (TF) and was detected serologically using specific lectins and antibody‐containing sera.Bovine fat globule membrane glycoproteins were the only source of Tn antigen. The Tn antigen was detected by anti‐Tn‐containing sera and by the lectins from Salvia sclarea and Helix pomatia as well as gas chromatographic detection of N‐acetylgalactosaminitol after alkaline borohydride treatment of the native glycoproteins. Human milk fat globule membrane glycoproteins showed the presence of blood group A substance by reaction with rabbit anti‐A serum.
The Organisation of Genes for Transfer RNA and Ribosomal RNA in Amoebae and Plasmodia of Physarum polycephalum
Authors: HALL Len , BRAUN Richard 
Published: March 2005

Using hybridisation techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 5.8‐S, 19‐S and 26‐S rRNA, 685 genes for 5‐S rRNA and 1050 genes for tRNA.
Hybridisation of these RNA species to both amoebal and plasmodial DNA fractionated on CsCl gradients reveal that the 5.8‐S, 19‐S and 26‐S rRNA genes are located at a satellite position (Q= 1.714 g/cm3) with respect to the main band of DNA, whereas 4‐S and 5‐S RNA genes are located exclusively in the main band of DNA (Q= 1.702 g/cm3).
This result was confirmed by demonstrating that only the 5.8‐S, 19‐S, and 26‐S rRNA species hybridise to purified plasmodial ribosomal DNA.
The 19‐S and 26‐S rRNA genes of amoebae are located on extrachromosomal DNA molecules of a discrete size (Mr= 38 × 106) with identical properties to plasmodial ribosomal DNA.
The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli
Authors: MÖLLER Klaus , RINKE Jutta , ROSS Alexander , BUDDLE Gerard , BRIMACOMBE Richard 
Published: March 2005

Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross‐linking reaction between protein and ribosomal RNA. Under the conditions described, about 10–15% of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein‐protein cross‐linking was observed. The RNA‐protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible.The proteins involved in the cross‐linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA‐protein complex. This showed that the individual ribosomal proteins were cross‐linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross‐linked in high yield.The formaldehyde‐treated subunits were subjected to a controlled hydrolysis with ribonuclease T1, and RNA fragments of known nucleotide sequence containing the cross‐linked proteins were isolated. Despite the reversibility of the cross‐linking reaction, sufficient cross‐linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments.In the 50‐S subunit, proteins L27 and L32 were found to be cross‐linked to the 18‐S RNA fragment which arises from the 3′ end of 23‐S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18‐S RNA and the 13‐S RNA fragment from the 5′ end of 23‐S RNA. In the 30‐S subunit, proteins S3, S5, S9 (11), S13, and to some extent S4, S7, S12 and S18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5′ end of 16‐S RNA. S4, S9 (11), S13, and to a lesser extent S7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3′ end of 16‐S RNA, but lacking the 3′‐terminal 150 nucleotides. Protein S21 was cross‐linked to 16‐S RNA, but was not found in either of the two RNA regions above.
Characterisation of RNA Fragments Obtained by Mild Nuclease Digestion of 30‐S Ribosomal Subunits from Escherichia coli
Authors: RINKE Jutta , ROSS Alexander , BRIMACOMBE Richard 
Published: March 2005

When Escherichia coli 30‐S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub‐fragments have therefore been examined by oligonucleotide analysis. Two well‐defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed.The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub‐fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′‐terminal 900 nucleotides of 16‐S RNA, corresponding to the well‐known 12‐S fragment. No long‐range interactions could be detected within this RNA region in these experiments.The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16‐S RNA, but lacking the 3′‐terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16‐S RNA, but section J (the 3′‐terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16‐S RNA is not involved in stable interactions with protein.
Puromycin and Some of Its Analogues: Conformational Properties in Solution
Authors: DE LEEUW Harry P. M. , DE JAGER Jenny R. , KOENERS Herman J. , VAN BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

The intramolecular conformation of puromycin in 2H2O solution at 25 °C and 65 °C was studied using vicinal coupling constants and chemical shifts obtained at 360 MHz. From vicinal coupling constants a 73% preference for the N‐type (C‐3′‐endo) ribose conformer is deduced. The reference compound, 6‐N‐dimethyl‐3′‐deoxy‐3′‐(acetamido)adenosine, displays even greater preference (80% N). Around the C‐4′– C‐5′ bond of the sugar residue and the C‐α–C‐β bond of the amino acid moiety the gauche+ rotamer (backbone notation) is preferred by 78% and 63% at 25 °C and 73% and 60% at 65 °C, respectively.From the proton magnetic resonance parameters of the equilibrating species 6‐N‐dimethyl‐3′‐(2′)‐O‐(methoxyacetyl)adenosine it is deduced that the gauche+ orientation of the exocyclic 5′‐CH2OH group is again preferred, but the nature and the position of the substituent exert an influence on the ribose conformational equilibrium. The change from 3′‐amide to 3′‐ester decreases the percentage of N‐type ribose to 34% in the latter whereas the 2′‐ester shows 55% preference for type N.Eight puromycin conformers are found to occur in significant amounts in solution, the most abundant conformer (36% at 25 °C and 31% at 65 °C) is the one identical with the solid‐state conformation of puromycin dihydrochloride. Calculated time‐averaged shielding values due to the phenyl ring of the amino acid moiety are in reasonable agreement with experiment and are dominated by the preferred conformer which is characterized by the proximity of the phenyl ring to the H‐4′, H‐5′ and H‐5” protons of the ribose moiety. The results substantiate the Remin and Shugar assignment of the C‐5′ methylene hydrogens in purine nucleosides. The possible biological significance of these findings is discussed.
On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast
Authors: VON DER HAAR Friedrich , CRAMER Friedrich , ECKSTEIN Fritz , STAHL Kurt‐Wilhelm 
Published: March 2005

Because of its chiralic α‐phosphorus atom adenosine 5′‐O‐(1‐thiotriphosphate) (ATPαS) exists in two diastereomeric forms, arbitrarily named (A) and (B).For phenylalanyl‐tRNA synthetase ATPαS (A) is a substrate whereas ATPαS (B) is neither a substrate nor an inhibitor. During the ATPαS (A)/PPi exchange reaction with phenylalanyl‐tRNA synthetase the configuration at the α‐phosphorus atom is retained. The mechanistic implications of these findings are discussed.Preliminary investigations with several other aminoacyl‐tRNA synthetases show that the stereochemical requirement with respect to the α‐phosphorus of ATP is not identical for all aminoacyl‐tRNA synthetases.