bluuebunny/crossref_metadata_2025_split huggingface dataset:
Macromolecular Complexes of Aminoacyl‐tRNA Synthetases from Eukaryotes
Authors: BREVET Annie , KELLERMANN Odile , TONETTI Hubert , WALLER Jean‐Pierre 
Published: March 2005

The gel‐filtration behaviour of a high‐molecular‐weight complex containing seven aminoacyl‐tRNA synthetases purified from sheep liver was examined on columns of 6% agarose (Bio‐Gel A‐5m). Evidence is provided for selective interaction of the complex with the agarose matrix.The binding capacity of agarose for the complex is dictated by the ionic strength of the equilibrating buffer: it is low but significant in 25 mM potassium phosphate buffer at pH 7.5 (ionic strength 0.068 M) and is considerably enhanced in 50 mM Tris‐HCl buffer at the same pH. In either case, raising the salt concentration leads to nearly complete recovery of enzyme activities.In a column equilibrated in 25 mM phosphate buffer, application of the complex at a protein concentration in large excess over the binding capacity of the gel leads to co‐elution of each of the seven aminoacyl‐tRNA synthetases as a unique, symmetrical peak of apparent molecular weight close to 106, with 80%, recovery of activities. Conversely, application of the complex at low protein concentration, in amounts equivalent to the binding capacity of the gel, leads to complete retention of the enzymes which may be recovered by raising the phosphate concentration to 0.2 M. It is further shown that application of the complex at low protein concentration on a column equilibrated with 0.2 M potassium phosphate buffer to prevent interaction with the matrix, leads to co‐elution of each of the aminoacyl‐tRNA synthetases as a unique peak of apparent molecular weight close to 106. This result attests to the remarkable stability of the complex, which fails to dissociate at high salt concentration, even in the diluted state.The above results are best interpreted as the reflection of ion‐exchange‐mediated interactions between the residual charged group on the agarose gel and the components of the high‐molecular‐weight complex. The preferential retention of the complex on agarose in ionic conditions (i. e. 25 mM phosphate or 50 mM Tris‐HCl buffers at pH 7.5) which ensure normal gel filtration behaviour of several marker proteins, may be ascribed to tighter binding of the complex due to multiple‐site interactions on account of its larger size.
Biocalcification by the Marine Alga Emiliania huxleyi (Lohmann) Kamptner
Authors: DE JONG Elisabeth , VAN RENS Lisette , WESTBROEK Peter , BOSCH Leendert 
Published: March 2005

Emiliania huxleyi (Lohmann) Kamptner, belonging to a group of marine unicellular algae, the Coccolithophoridae, possesses a cell wall containing calcified structures called coccoliths. These coccoliths contain a water‐soluble acid polysaccharide. The polysaccharide contains ester sulphate and uronic acid groups and binds Ca2+ preferentially from a medium also containing Na+ and Mg2+. It is thought to perform a regulatory function in the calcification process. In the present paper we describe a series of studies in vivo intended to give a preliminary characterization of a number of metabolic steps leading to coccolith formation. In view of the putative role of the acid polysaccharide in coccolith synthesis, we have concentrated on the incorporation of polysaccharide precursors by calcifying cells. As a second approach we investigated the differences between calcifying cells and cells of the same species that have lost the capability to produce coccoliths (non‐calcifying cells).Calcifying cells were offered radioactive calcium, bicarbonate, galactose and sulphate in the light. Most of the calcium label and a small part of the label of galactose and bicarbonate was incorporated into extracellular coccoliths. The label of galactose was detected in at least seven of the constituent monosaccharides of the coccolith‐associated polysaccharide. Radioactive sulphate was also incorporated into the extracellular coccolith polysaccharide. In contrast to the label of bicarbonate and galactose that of calcium reached a steady‐state concentration intracellularly within 2 h. A technique was developed for the isolation of intracellular CaCO3, presumably coccoliths in statu nascendi. About 10% of the label of bicarbonate and galactose was incorporated into an intracellular acid polysaccharide, resembling the coccolith‐associated macromolecule. Non‐calcifying cells fail to take up Ca2+ and galactose. We demonstrated that non‐calcifying cells produce an acid polysaccharide, resembling the coccolith‐associated macromolecule. The former polysaccharide is shed into the surrounding medium and can be labelled by offering the non‐calcifying cells radioactive bicarbonate.
NAD‐Dependent Formate Dehydrogenase from Methylotrophic Bacterium, Strain 1
Authors: EGOROV Aleksey M. , AVILOVA Tatyana V. , DIKOV Michael M. , POPOV Vladimir O. , RODIONOV Yuriy V. , BEREZIN Ilia V. 
Published: March 2005

1. NAD‐dependent formate dehydrogenase was isolated from gram‐negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion‐exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40 %.2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultra‐centrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2‐mercaptoethanol.3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80000.4. Amino acid composition and some other physico‐chemical properties of the enzyme were studied.5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37°C).6. Formate dehydrogenase was rapidly inactivated in the absence of –SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2‐mercaptoethanol or EDTA.
Electron Paramagnetic Resonance of the Copper in Dopamine β‐Monooxygenase
Authors: SKOTLAND Tore , PETERSSON Leif , BÄCKSTRÖM Dan , LJONES Torbjørn , FLATMARK Torgeir , EHRENBERG Anders 
Published: March 2005

The water‐soluble form of dopamine β‐monooxygenase from bovine adrenal medulla was studied. Addition of excess CuSO4 to purified enzyme preparations followed by extensive ultrafiltration against copper‐free buffer at pH 7.0, gave preparations with about four copper atoms per enzyme tetramer of Mr 290000. The enzyme‐bound copper was shown by the rapid‐freeze technique and electron paramagnetic resonance (EPR) to be reduced by ascorbate at a rate which was faster than the overall catalytic rate; about 10% of the copper was oxidized to Cu(II) during steady‐state catalysis in the presence of excess ascorbate. These results support an electron‐transfer function of the enzyme‐bound copper during catalysis and indicate that the reduction by ascorbate is not the rate‐limiting step.The enzyme‐bound copper was rapidly chelated by EDTA at pH 7.0, and the apoenzyme thus obtained after dialysis revealed no EPR‐detectable copper. Addition of CuSO4 to the apoenzyme gave an EPR spectrum similar to that of the native enzyme, and the apoenzyme was fully reactivated by the optimal concentration of CuSO4 in less than 2 s.
Locust Collagen: Morphological and Biochemical Characterization
Authors: ASHHURST Doreen E. , BAILEY Allen J. 
Published: March 2005

Segment‐long‐spacing crystallites and reconstituted fibrils have been made from collagen extracted from the ejaculatory duct of the adult male locust, Locusta migratoria. These show the same banding pattern after positive staining as segment‐long‐spacing crystallites and fibrils made from mammalian type I collagen. Native fibrils show the same periodic pattern as type I fibrils, but it is not so distinct.Biochemical analysis of pepsin‐digested locust collagen shows that there are two collagenous components. The α chains of the major component are similar to mammalian α1 (I) chains, except that the number of hydroxylysine residues is elevated and the CNBr peptides differ. There are no α2 chains; hence this locust collagen molecule is an α1 trimer. The second component, which is present in only minute quantities, may be a type IV basement membrane collagen.It is concluded that the fibrous collagen molecule of the locust is very similar to that of mammalian type I trimers and to those of other invertebrates which have been examined.
Biosynthèse de cellulose bactérienne à partir de d‐glucose uniformément enrichi en 13C
Authors: GAGNAIRE Didier Y. , TARAVEL François R. 
Published: March 2005

Biosynthesis of Bacterial Cellulose from d‐Glucose Uniformly Enriched in 13C As a typical illustration of the use of a statistically‐enriched 13C precursor for nuclear‐magnetic‐resonance (NMR) studies of biosynthetic mechanisms, the following example is described.A sample of cellulose was synthesized by Acetobacter xylinum from a 50–50 mixture of d‐glucose and statistically enriched d‐[13C]glucose (87%13C). Each carbon atom of the cellulose is equally enriched in 13C (35%), as seen from the NMR spectrum of the acetate. A precise analysis of this product was performed by chemical transformation into a small molecule (3‐O‐acetyl‐1,2;5,6‐di‐O‐isopropylidene‐α‐d‐glucofuranose) whose 1J(13C‐13C) coupling constants provide for a direct determination of the four populations of molecules with a 13C at the Q position (i= 2, 3, 4, 5) having as neighbours (Ci−1 and Ci+1)12C or 13C.A general statistical treatment of such problems is proposed, with the determination of two kinds of parameters.For two consecutive carbons, the terms Pi,i+1 give the probability of both carbons Ci and Ci+1 in the final product coming from the same precursor molecule. The experimental values are: P1,2= 0.89, P2,3= 0.60, P3,4= 0.26, P4,5= 0.95, and P5,6= 0.93.For three consecutive carbons, in some favourable cases, a correction term Ci,i+2 is introduced, to explain experimental deviations of populations from those calculated with two independant probabilities Pi,i+1 and Pi+1,i+2 In the example described here, two terms can be determined: C1,3= 0.066, C2,3= 0.44.Seven values of P and C are obtained in a single experiment. These values allow for a correct interpretation of other experiments that are described, such as biosynthesis where pure statistically 13C‐enriched glucose is used as precursor; experiments with specifically labelled glucose ([1‐13C] and [6‐13C]); and the global isotopic composition of all products, as measured by mass spectrometry.The biosynthetic interpretation is done in terms of the following pathways: (i) direct incorporation, (ii) the pentose phosphate cycle (giving CO2, glyceraldehyde 3‐phosphate and glucose 6‐phosphate), (iii) the Entner‐Doudoroff pathway (giving pyruvate and glyceraldehyde 3‐phosphate), (iv) triose recombination (giving pyruvate and glucose 6‐phosphate).The interpretation of the seven experimental terms P and C does not allow for constant percentages for these pathways during the biosynthesis, but indicates the following values for the whole process: (i) 23% of the glucose molecules in cellulose come from an unmodified molecule of glucose; (ii) 26% have been involved at least once in the pentose cycle, and 10 at least twice; (iii) 30% of the glucose molecules come from triose recombination.
Escherichia coli Murein Transglycosylase
Authors: KUSSER Wolfgang , SCHWARZ Uli 
Published: March 2005

Escherichia coli murein transglycosylase, a potential autolysin which splits the sugar chains of the murein sacculus, was rapidly purified from a crude cell extract by sequential chromatography on columns of blue Sepharose and poly(U)‐Sepharose. In accordance with the binding to blue Sepharose and poly(U)‐Sepharose, the transglycosylase is inhibited by Cibacron blue F3G‐A, the affinity ligand of blue Sepharose, and also by polynucleotides, the latter, however, with varying efficiency. Among the polynucleotides tested, single‐stranded DNA was found to be one of the most potent inhibitors.When bound to a blue Sepharose column, transglycosylase could be displaced from the column with single‐stranded DNA. Taken together, these results point to a polynucleotide binding area on the transglycosylase molecule.Some aspects of the blue Sepharose affinity chromatography and the possible biological significance of the transglycosylase are discussed.
Binding Kinetics of 4‐Methylumbelliferyl α‐Mannobioside to Concanavalin A by Fluorescence Stopped‐Flow Measurements
Authors: VAN LANDSCHOOT Anita , LOONTIENS Frank G. , CLEGG Robert M. , JOVIN Thomas M. 
Published: March 2005

The stopped‐flow binding kinetics of concanavalin A with fluorescently labelled (α 1 → 2)‐linked manno‐oligosaccharides are characterized by signal changes which are monophasic for 4‐methyl‐umbelliferyl α‐d‐mannopyranoside (I), biphasic for 4‐methylumbelliferyl α‐mannobioside(II) and triphasic for 4‐methylumbelliferyl α‐mannotrioside (III). The results indicate that the carbohydrate‐binding site interacts specifically with a single mannopyranosyl group, resulting in as many possible complexes as there are mannopyranosyl groups in the oligosaccharide chain.Pseudo‐first‐order kinetics with II and concanavalin A in the range 6–334 μM yield a faster time constant (110–37 ms) with a saturable amplitude and a slower one (3.9–0.29s) with an amplitude which reaches a peak at 30 μM. The concentration dependencies of the two apparent rate constants and the corresponding amplitudes are compatible with two equivalent mechanisms for which the reaction‐rate parameters are calculated. Each involves two complexes with spectroscopic parameters which are dependent upon the mechanism. The first mechanism consists of binding followed by an isomerisation, interpreted as sliding of the internal mannopyranosyl residue to bind preferentially via the terminal residue. In the second mechanism, these two binding modes would alternate; here, the binding via the terminal residue is preferred at equilibrium by a factor of 68; both association‐rate parameters are comparable and similar to those for I or simple glycosides. Consistent results were obtained by temperature‐jump relaxation kinetics.
Functional Role of Ribosylthymine in Transfer RNA
Authors: DINGERMANN Theodor , PISTEL Friedbert , KERSTEN Helga 
Published: March 2005

In some eucaryotes tRNA species occur containing exclusively uridine or ribosylthymine at position 54. In other eucaryotic tRNA species varying ratios of uridine to ribosylthymine have been found. To elucidate whether variation in the degree of U54 methylation in eucaryotic tRNA plays a functional role in protein synthesis, polysomal and overal cellular tRNAs in the lower eucaryote Dictyostelium discoideum were characterized during vegetative growth and the early developmental preaggregation stage.The nucleoside composition of the tRNAs was determined and U54‐containing tRNA species were detected after methylation in vitro with the rT‐forming enzyme from Escherichia coli. The following results were obtained. (a) During vegetative growth, 85% of the tRNAs present on polysomes contain rT at position 54, whereas tRNA species containing U54 accumulate in non‐polysomal cell fractions. (b) Overall cellular and polysomal tRNAs from the preaggregation stage are less modified with respect to rT54 than those from the vegetative growth stage; in addition pseudo‐uridine (Ψ) and 5‐methylcytidine (m5c) are partially lacking at tRNA positions not yet identified. (c) The rT‐containing tRNAs predominate on polysomes also during preaggregation; however, only 65% of the polysomal tRNAs contain ribosylthymine.The results demonstrate that in D. discoideum, the major class tRNAs utilized for protein synthesis includes molecular species in which a U54 residue has been methylated to rT. The ratio of rT54 to U54 in these tRNAs declines at a physiological stage at which the rate of protein synthesis is reduced. Since in vitro an eucaryotic U54‐containing tRNA of this class translates mRNA less efficiently than its rT‐containing counterpart [K. B. Marcu and B. S. Dudock 1976 Nature (Lond.) 261, 159–162], it is assumed that these U54‐containing tRNAs are rate‐limiting in the elongation of peptide chains in eucaryotes.
Chloroplast Phosphoproteins. Evidence for a Thylakoid‐Bound Phosphoprotein Phosphatase
Authors: BENNETT John 
Published: March 2005

Isolated intact pea (Pisum sativum) chloroplasts incorporate [32P]orthophosphate into several thylakoid polypeptides in the light. Transfer of the labelled chloroplasts to darkness results in rapid dephosphorylation of the polypeptides. The most rapidly dephosphorylated phosphoproteins are the 26000‐Mr doublet derived from the light‐harvesting chlorophyll a/b binding complex.
Incubation of isolated 32P‐labelled thylakoids in buffer in the absence of stromal components also results in rapid protein dephosphorylation. Again, the most rapidly dephosphorylated phosphoproteins are the 26000‐Mr light‐harvesting doublet. Dephosphorylation of all thylakoid phosphoproteins is accelerated by addition of up to 10 mM MgCl2.
The enzyme responsible for dephosphorylation is a phosphatase rather than a phosphotransferase or the thylakoid protein kinase acting in reverse. The enzyme is specifically and totally inhibited by NaF and does not require phosphoryl group acceptors such as ADP. Unlike the protein kinase, the phosphatase is indifferent to light and the electron transport inhibitor 3(3,4‐dichlorophenyl)‐1,1‐dimethylurea.
The phosphorylated regions of the thylakoid phosphoproteins protrude from the outer surface of the membrane and are removed by trypsin treatment.
Cell‐Free Translation and Regulation of Candida lipolytica Acetyl‐Coenzyme‐A Carboxylase Messenger RNA
Authors: HORIKAWA Saburo , KAMIRYO Tatsuyuki , NAKANISHI Shigetada , NUMA Shosaku 
Published: March 2005

Messenger RNA from Candida lipolytica directs the synthesis of complete acetyl‐coenzyme‐A carboxylase in the mRNA‐dependent reticulocyte lysate cell‐free translation system. The identity of the translation product is evidenced by the following results: first, it is immunoprecipitated with antibody to acetyl‐CoA carboxylase and competes with authentic acetyl‐CoA carboxylase for binding to the antibody; secondly, it co‐migrates with authentic acetyl‐CoA carboxylase (Mr 230000) upon dodecylsulfate/polyacrylamide gel electrophoresis; finally, the peptide fragments formed by its partial proteolysis with papain or α‐chymotrypsin are identical with those formed from authentic acetyl‐CoA carboxylase.With the use of this assay system, it has been demonstrated that the level of acetyl‐CoA carboxylase mRNA activity in C. lipolytica cells decreases with increasing concentrations of oleic acid in culture medium and that the changes in the mRNA activity parallel those in the cellular level of acetyl‐CoA carboxylase. This finding, in conjunction with our previous study, indicates that the diminished synthesis of acetyl‐CoA carboxylase in cells grown in the presence of fatty acid is due to a reduced level of the mRNA coding for the enzyme.
Activities of 3‐Hydroxybutyrate Dehydrogenase, 3‐Oxoacid CoA‐Transferase and Acetoacetyl‐CoA Thiolase in Relation to Ketone‐Body Utilisation in Muscles from Vertebrates and Invertebrates
Authors: BEIS Androniti , ZAMMIT Victor A. , NEWSHOLME Eric A. 
Published: March 2005

The activities of 3‐hydroxybutyrate dehydrogenase were non‐detectable in muscles of invertebrates and marine teleost fish; activities were found in muscles of amphibia, reptiles and mammals and also in an elasmobranch fish. Muscles were classified into three groups according to the activities of 3‐oxoacid CoA‐transferase: muscles with very low activities (> 0.01 μmol × min−1× g−1) which obtain energy for contraction from anaerobic glycolysis; muscles with low activities (< 0.01 >5 μmol × min−1× g−1) which include insect flight muscles, muscles of other invertebrates and skeletal muscles of higher vertebrates; muscles with high activities of 3‐oxoacid CoA‐transferase (< 5 μmol × min−1× g−1) which are characterised by continuous mechanical activity for long periods of time, e.g. heart, diaphragm, postural and some smooth muscles of mammals.
It is suggested that ketone bodies may be important fuels for muscles in the very low and low activity groups during starvation, when the muscle is at rest. The muscles in the high activity group may use ketone bodies when they are available in the blood to provide energy for mechanical activity. Since these muscles provide a continuous vital physiological function, they must always be provided with a fuel for respiration and, in a similar manner to brain, they may utilise either glucose or ketone bodies.
Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein
Authors: JÖRNVALL Hans , PHILIPSON Lennart 
Published: March 2005

The sulfhydryl group of one cysteine residue in the adenovirus hexon protein is accessible to alkylation in the absence of denaturating agents. This residue was [14C]carboxymethylated and characterized in peptides after proteolytic treatments. It was identified in two different types of digest and corresponded to the cysteine residue most accessible to oxidation. It is located close to residue 670 in the tentative amino acid sequence of the entire polypeptide.Trypsin has a highly limited action on the native hexon protein. Only three peptide bonds are fully susceptible to cleavage. These were found to occur in the N‐terminal third of the protein, and were characterized as cleavages close to positions 150, 180 and 320 in the tentative structure of the polypeptide. Trypsin therefore liberates four fragments in major yield, with approximate Mr of 3000, 15000, 17000 and 78000. The smallest fragment corresponds to a highly acidic region. A limited, but less restricted susceptibility of hexon to chymotryptic cleavage was also found, and four major sites were characterized.The cysteine labelling and the proteolytic treatments define accessible regions in the native conformation of hexon. Together with previously assigned residue distributions, they suggest the subdivision of the entire polypeptide chain into three approximately equal parts, with accessible sites in the border regions, as well as in the middle of at least the N‐terminal third. One model compatible with all data is presented.
ADP‐Ribosylation of Nuclear Proteins in Normal Lymphocytes and in Low‐Grade Malignant Non‐Hodgkin Lymphoma Cells
Authors: WIELCKENS Klaus , GARBRECHT Manfred , KITTLER Margrid , HILZ Helmuth 
Published: March 2005

Normal lymphocytes and lymphocytes from patients with low‐grade malignant non‐Hodgkin lymphoma were isolated from blood by a Percoll gradient procedure. Absence of cell proliferation in both cell types was indicated by very low [3H]thymidine incorporation rates.Determination of endogenous protein‐bound single ADP‐ribose residues by a radioimmunoassay revealed that the leukemic cells had 2.5‐times lower levels of the NH2OH‐sensitive and a 4‐fold lower amount of NH2OH‐resistant ADP‐ribose · protein conjugate subfractions, respectively, than normal lymphocytes.By contrast, ‘total’ ADP‐ribose transferase activity, as measured in homogenates or permeabilized cells in the presence of DNase, was two‐times higher in leukemic cells, whereas activity determined in permeabilized cells in the absence of added DNase was practically identical in both cell types.The apparent discrepancy between ADP‐ribose transferase activity and endogenous levels of protein‐bound single ADP‐ribose residues may be explained in part by an enzyme inhibitor present in normal human lymphocytes.NAD + NADH levels were decreased 2.5‐fold in the leukemic cells. This decrease, however, does not explain the reduced levels of mono(ADP‐ribose) · protein conjugates since the ratio of protein‐bound single ADP‐ribose residues to NAD is distinctly different in leukemic lymphocytes compared to normal lymphocytes.