bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA
Authors: ARNEMANN Jutta , HEINS Bärbel , BEATO Miguel 
Published: March 2005

Uteroglobin, a progesterone‐induced uterine protein of the rabbit, is synthesized in cell‐free systems as a precursor containing 21 additional amino‐acids at its N‐terminal end. The mRNA for pre‐uteroglobin has been purified from the membrane‐bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of r0t(0.01 M · s) and digestion of the non‐hybridized cDNA by S1 nuclease.A comparison of the hybridization kinetics of the pre‐uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre‐uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamide. The melting temperature of the hybrids of pre‐uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA has been used to quantify pre‐uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobin‐synthesizing activity, there is a dramatic increase in the uterine content of pre‐uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on a DNA basis there is a 50–100‐fold increase in the cellular content of pre‐uteroglobin mRNA following hormonal treatment.
Kinetic Mechanism of NADH‐Dependent Glutamate Synthase from Lupin Nodules
Authors: BOLAND Michael J. 
Published: March 2005

From initial‐rate studies, a partially random kinetic mechanism has been deduced for NADH‐dependent glutamate synthase from lupin nodules. The mechanism involves compulsory binding of NADH as first substrate, followed by random‐order binding of glutamine and 2‐oxoglutarate. Patterns of inhibition by glutamate substantiate the mechanism. Dithionite was incapable of acting as an alternative reducing substrate although it is known to reduce the flavine groups of the enzyme. The implications of these results are discussed.Published rate equations for this type of mechanism were found to be unsatisfactory for this enzyme and suitable new equations are produced. These equations should have general application where the obligatory first substrate binds very tightly.
Metabolism of Isolated Kidney Tubules
Authors: GUDER Walter G. , WIRTHENSOHN Gabriele , Pürschel Susanne , Gerl Margit 
Published: March 2005

Kidney‐cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L‐lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations.All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process.Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75 % of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased.The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.
Two Distinct Poly(A) Polymerases Isolated from the Cytoplasm of Ehrlich Ascites Tumour Cells
Authors: AVRAMOVA Zoya V. , MILCHEV Georgui I. , HADJIOLOV Asen A. 
Published: March 2005

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE‐cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+‐dependent poly(A) polymerase (ATP: RNA adenylyltransferase) and (b) a ribosome‐associated enzyme defined tentatively as ATP(UTP) :RNA nucleotidyltransferase.The cytosol poly(A) polymerase is strictly Mn2+‐dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, Poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin.The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10–20 mM Mg2+. The enzyme uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.
Primer Specificity of Ribosome‐Associated Poly(A) Polymerase from Ehrlich Ascites Tumour Cells
Authors: MILCHEV Georgui I. , AVRAMOVA Zoya V. , HADJIOLOV Asen A. 
Published: March 2005

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP):RNA ucleotidyltrasferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50% of the [3H]AMP label is in poly(A)‐containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8–30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10–S zone. The poly(A) polymerase catalyzes the addition of 12–18 adenylate residues to pre‐existing mRNA poly(A) sequences of 40–160 residues. The [3H]AMP incorporated into poly(A)‐lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4‐S RNA. In the purified enzyme system, specificity towards poly(A)‐containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)‐cellulose. The bulk of the product is in 18‐S rRNA and heterogeneous small molecular weight RNA.We conclude that the ribosome‐associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)‐containing mRNA in vivo.
Fluorimetric Study of the Complex between Yeast Phenylalanyl‐tRNA Synthetase and tRNAPhe
Authors: LEFEVRE Jean‐François , EHRLICH Ricardo , REMY Pierre 
Published: March 2005

The variations of several spectroscopic properties of yeast tRNAPhe and phenylalanyl‐tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions.In all cases, for the tRNAPhe‐enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.
Chain Inequivalence in Bovine Methemoglobin
Authors: ILAN Yael A. , ILAN Yigal , CHEVION Mordechai , CZAPSKI Gidon 
Published: March 2005

Using pulse radiolysis, a single heme in the tetramer of bovine methemoglobin was reduced within a few microseconds to the ferro state, producing a valence intermediate. The kinetics of oxygen binding to the valence intermediate as well as the re‐oxidation of the ferro‐heme to the ferric state were studied as a function of pH.The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectively). A sigmoidal curve representing a transition between the two states as a function of pH was derived. Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant.The reaction between the valence intermediate and ferricyanide at pH 7.75 (R state) consisted of two (about) equal contributions (k1= 23 × 104M−1 s−1; k2= 2.1 × 104 M−1 s−1) attributed to the β and α subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was observed (k1= 69 × 104 M−1 s−1; k2= 3.7 × 104 M−1 s−1), indicating chain inequivalences both in the T and the R states of methemoglobin.In the presence of inositol hexakisphosphate the T → R transition, as monitored by oxygenation of the valence intermediate, was shifted up to a higher pH by about 0.35. Yet similar rate constants exhibiting similar chain inequivalences have been measured.
Arginyl‐Transfer Ribonucleic Acid Synthetase of Bacillus stearothermophilus. Purification and Kinetic Analysis
Authors: GODEAU Jean‐Marie 
Published: March 2005

Arginyl‐tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified 880‐fold to apparent homogeneity as demonstrated by electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight is 59000 as confirmed by Sephadex G‐100 and by sucrose gradient ultracentrifugation. The enzyme is monomeric, no subunits were detected. Its cognate tRNA induces an apparent increase in molecular weight suggesting the dimerisation of the enzyme. Nevertheless, it is not obvious that the enzyme dimer forms prior to the aminoacylation reaction catalysed by the enzyme. An ATPase activity was found associated to the synthetase but can be neglected because the ATP consumption is too low for hampering the arginyl‐tRNA synthetase activity.The order of addition of substrates and release of products has been studied by measurements of initial velocity, product inhibition and dead‐end inhibition. The nature of the kinetic patterns indicates that the aminoacylation reaction conforms to the classical concept of the mechanism which includes the formation of an enzyme‐bound aminoacyl‐adenylate as an intermediate in the first step followed by transfer of the amino acid to tRNA. The first partial reaction, measured by the ATP‐32PPi exchange or AMP synthesis in the presence of ATP and arginine, requires tRNA, which is consistent with the model in which tRNAArg is an activator of the arginyladenylate synthesis.
Interaction between Major Outer Membrane Protein (O‐8) and Lipopolysaccharide in Escherichia coli K12
Authors: YAMADA Hisami , MIZUSHIMA Shoji 
Published: March 2005

An ordered hexagonal lattice structure with a lattice constant of about 7nm was reconstituted from outer membrane protein O‐8 and/or O‐9 and lipopolysaccharide [H. Yamada and S. Mizushima, J. Bacteriol. 135, 1024–1031 (1978)]. Using this reconstitution system, the interaction between O‐8 and lipopolysaccharide was studied and the following results were obtained.At least one lipopolysaccharide molecule per one O‐8 trimer was required for the hexagonal lattice formation. The lattice constant of the hexagonal lattice formed under the conditions was about 6.5 nm. In the presence of a larger amount of lipopolysaccharide, the lattice constant increased to 7.5 nm, being almost the same as that observed in the dodecylsulfate‐treated cell envelope.The lattice constant observed with the heptoseless lipopolysaccharide was also 6.5 nm when the lipopolysaccharide/O‐8 ratio was low. Although the increase of the lattice constant was observed upon further addition of the lipopolysaccharide, it was appreciably smaller than that with the wild‐type lipopolysaccharide.A hexagonal lattice was also formed when lipopolysaccharide was replaced by an equivalent amount of lipid A or even fatty acids. The lattice constant was the same as that with the wild‐type lipopolysaccharide when the lipid/protein ratio was low. However, contrary to the case of lipopolysaccharide, the lattice constant remained almost unchanged even when a larger amount of either lipid A or fatty acid was added to the reconstitution system. A slight increase in the lattice constant was observed only when an extremely large amount of lipid A was added to the reconstitution system.These results suggest that (1) both the lipid A moiety and the polysaccharide moiety of lipopolysaccharide participate in the interaction with the O‐8 trimer, (2) in the lipid A moiety the fatty acid region primarily participates in the hexagonal arrangement of the O‐8 trimer and the minimum requirement of lipopolysaccharide for the lattice formation is most probably one lipopolysaccharide molecule per O‐8 trimer and (3) the polysaccharide moiety, probably both the 3‐deoxy‐d‐manno‐octulosonate region and the distal end region including heptose, are involved in an interaction of lipopolysaccharide with the O‐8 trimer to give a proper lattice conformation.
Loops in Viroids
Authors: WILD Ursula , RAMM Karla , SÄNGER Heinz Ludwig , RIESNER Detlev 
Published: March 2005

The structure of the potato spindle tuber viroid (PSTV) was investigated by binding of specific tRNAs to the molecule. Instead of oligonucleotides tRNAs had to be used for those studies because of their higher binding constants and the easier methods of monitoring the extent of binding. Complex formation was detected by analytical ultracentrifugation and by optical titrations. Buffer conditions were 0.05 M sodium cacodylate, 0.01 M MgCl2, pH 6.8 in the optical titrations, and 0.1 M NaCl was added in the ultracentrifugation experiments.From sedimentation analysis at 10°C following results were obtained: tRNALys from Escherichia coli, tRNAphe, tRNAAsp, and tRNASer from yeast bind to PSTV with binding constants in the order of 105 M−1. No binding could be detected with tRNAfMet and tRNA1Val from E. coli.In spectrophotometric titrations with tRNA2Glu and tRNALys from E. coli the decrease in the absorption of mnm5s2U at 300 nm due to the binding was observed. 1 mol tRNA2Glu/mol PSTV binds with a constant of 5.3 × 105 M−1 at 10°C; 3 mol tRNALys/mol PSTV bind with an average constant of 7.0 × 104 M−1 at 20°C.In fluorimetric titrations with tRNAphe from yeast the quenching of the fluorescence intensity of the wye base due to the binding was monitored. 2 mol tRNAphe/mol PSTV bind with an average constant of 7.5 × 105M−1 at 10°C.From the experimental results the following conclusions can be drawn. The anticodons of several specific tRNAs bind to complementary sequences in certain loop regions of PSTV. The loop regions determined by this method are in accordance with the nucleotide sequence and the secondary structure model of PSTV as published recently [2,4]. The bases in the internal loops must have a sufficient degree of flexibility to assume a spatial arrangement which is optimal for base‐pairing with other RNAs. The free accessibility of loops distributed over the entire PSTV molecule clearly demonstrates the absence of tertiary structure folding in PSTV.
Further Characterization of the Phosphate Moiety of the Adenovirus Type 2 DNA‐Binding Protein
Authors: LINNÉ Tommy , PHILIPSON Lennart 
Published: March 2005

The adenovirus type 2 DNA‐binding protein is phosphorylated. Alkaline phosphatase treatment removes phosphate groups resulting in a decrease in molecular weight from 72000 to 70000. The dephosphorylated protein binds to single‐stranded and double‐stranded DNA as well as the phosphorylated protein does. Controlled chymotrypsin treatment cleaves the DNA‐binding protein into two subspecies of Mr about 45000 and 25000. The 45000‐Mr polypeptide contains most of the methionine residues but no phosphate and binds to DNA. The 25000‐Mr polypeptide contains all the phosphate groups and shows no binding to DNA. Isoelectric focusing gels show heterogeneity of the DNA‐binding protein and 15 subspecies with different charges can be observed after partial dephosphorylation by alkaline phosphatase. After extensive dephosphorylation two or three basic species with a molecular weight around 70000 are observed. Quantitative immunoprecipitation from cells labeled to equilibrium with inorganic 32PO4 gives a molar ratio of phosphate to protein of 4–7 and direct chemical determination of the phosphate residues yields 4 mol Pi/mol protein. These results suggest that there exist subspecies of the protein moiety of the adenovirus DNA‐binding protein.The DNA‐binding protein isolated from infected cells after a short ‘pulse’ of [35S]methionine has a molecular weight which corresponds to that of the dephosphorylated protein. After a ‘chase’ period the molecular weight increases to 72000, but alkaline phosphatase treatment converts it to a species with the same molecular weight as the newly synthesized DNA‐binding protein, indicating that the modification of the protein is due to phosphorylation.
Transbilayer Distribution and Mobility of Phosphatidylcholine in Intact Erythrocyte Membranes
Authors: VAN MEER Gerrit , POORTHUIS Ben J. H. M. , WIRTZ Karel W. A. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M. 
Published: March 2005

The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine‐specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles and rat liver microsomes, much higher concentrations of exchange protein were required in the case of intact red blood cells and microsomes.
In human erythrocytes, 75% of the phosphatidylcholine was available for exchange within 2 h at 37°C. No additional exchange was observed during the next 2 h, indicating slow, if any, trans‐bilayer movement of the residual phosphatidylcholine.
In rat erythrocytes 50–60% of the phosphatidylcholine was readily available for the exchange proteins. The residual phosphatidylcholine was exchanged at a much lower rate with a half time for equilibration of 7 h.
These results confirm in an independent way the asymmetric distribution of phosphatidylcholine over the membrane of human and rat erythrocytes as well as the occurrence of a slow transbilayer movement of this lipid in rat erythrocytes.
31P Nuclear‐Magnetic‐Resonance Studies of Energy Metabolism in Tissue from the Marine Invertebrate Tapes watlingi
Authors: BARROW Kevin D. , JAMIESON Dana D. , NORTON Raymond S. 
Published: March 2005

31P nuclear magnetic resonance spectra of tissue from the marine bivalve mollusc Tapes watlingi are presented. Spectra from foot muscle, adductor muscle and hearts contain resonances from the adenine nucleotides, inorganic phosphate and a phosphagen which is shown to be arginine phosphate. The intracellular pH of the tissue, measured from the chemical shift of the inorganic phosphate resonance, is 7.2, which is significantly lower than that of sea water, viz. 8.1. The importance of a correct choice of solution conditions for determining a correlation curve of chemical shift versus pH for tissue from marine organisms is emphasized. Spectra of perchloric acid extracts of muscle are used to determine levels of the major phosphorus‐containing metabolites in the tissue.The time courses of changes in the phosphorus‐containing metabolites of foot and adductor muscle are described. Under anaerobic conditions the sequence of events is similar to that observed in vertebrate skeletal muscle, but the overall rate of decay is much slower, e.g. arginine phosphate is present in foot muscle for almost 20 h at its physiological temperature. Oxygenation of the incubation medium prolongs the survival time of the tissue. Treatment with iodoacetamide and sodium azide under anaerobic conditions reduces the lifetime and changes the pattern of decay such that ATP begins to decay before the phosphagen is consumed, and most of the phosphate accumulates in sugar phosphates, mainly fructose 1,6‐biphosphate.The effect of low salinity stress is also examined. Spectra of muscle from animals exposed to 50% and 75% normal sea water for periods of about four weeks are essentially identical with those of normal animals. Furthermore, the decay of intact muscle incubated in 50% sea water is similar to that in normal sea water.
Messenger RNA Coding for Phenylalanine Ammonia‐Lyase
Authors: RAGG Hermann , HAHLBROCK Klaus 
Published: March 2005

The mRNA coding for phenylalanine ammonia‐lyase was partially purified from irradiated cell suspension cultures of parsley (Petroselinum hortense). The product of cell‐free translation of the mRNA in a reticulocyte lysate was isolated by immunoprecipitation and compared with the native enzyme subunit. Evidence for the identity, or at least a great similarity, of both was provided by tryptic‐peptide and gel‐electrophoretic analyses.Under partially denaturing conditions, phenylalanine ammonia‐lyase mRNA sedimented as a 20–21‐S molecule in a sucrose gradient and had an apparent molecular weight of about 1.05 × 106on a polyacrylamide gel. Approximately two‐thirds of the polynucleotide sequence of the mRNA were estimated to be required as coding sequence for the enzyme. We suggest that phenylalanine ammonia‐lyase mRNA is unlikely to code for more than one of three coordinately induced enzymes.
The Activation of Brain Adenylate Cyclase and Brain Cyclic‐Nucleotide Phosphodiesterase by Seven Calmodulin Derivatives
Authors: THIRY Philippe , VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , RATHE Jean , CHRISTOPHE Jean 
Published: March 2005

A comparison has been made of the ability of seven calmodulin derivatives to displace 125I‐labeled calmodulin and to activate adenylate cyclase in a brain particulate fraction. The activation of brain‐soluble cyclic‐nucleotide Phosphodiesterase by the same calmodulin derivatives was examined in parallel.
In general, the dose for half‐maximal inhibition of 125I‐labeled calmodulin binding and the apparent Km of adenylate cyclase activation were comparable in brain membranes. These concentrations were 20–40‐times higher than the corresponding apparent Km values of activation of cyclic‐nucleotide Phosphodiesterase.
Modifying the single histidine residue or both tyrosine residues exerted no influence on the biological properties of calmodulin. The carboxymethylation of two methionine residues or the amidation of several carboxyl groups reduced the activation properties of calmodulin on adenylate cyclase and cyclic‐nucleotide Phosphodiesterase. Altering seven lysine or four arginine residues resulted in two proteins whose activation properties on adenylate cyclase and Phosphodiesterase had been modified in a way suggesting that lysine and arginine residues play distinct roles in the interaction of native calmodulin with each enzyme.