bluuebunny/crossref_metadata_2025_split huggingface dataset:
Initial Stages of the Thermal Unfolding of Yeast Phenylalanine Transfer RNA as Studied by Chemical Modification: The Effect of Magnesium
Authors: RHODES Daniela 
Published: March 2005

The early stages of thermal unfolding of the tertiary structure of yeast tRNAphe have been followed, in the presence and absence of Mg2+, by measuring changes in the chemical accessibility of the bases uracil and guanine. The reagent used in these studies is 1‐cyclohexyl 3‐[2‐morpholino(4)‐ethyl]carbodiimide methotosylate. 32P‐labelled tRNA was used so that the points of modification could be examined with ribonuclease digestion and established fingerprinting techniques.Two regions of protection by Mg2+ have been found. One is within the oligonucleotide U8‐A‐m2G10 and the other is in the vicinity of residue U‐59.The tertiary interactions and the D stem are the most readily melted parts of the tertiary structure. In the absence of Mg2+ the region of U‐59 is the first part of the tertiary structure to become accessible to the reagent. This is closely followed by the opening up of the ‘wobble’ G · U base pair in the aminoacyl stem. Most of the triple interactions in the augmented D helix are also disrupted early in the melting. The region of intricate interactions between the invariant G‐G part of the D loop and the T‐Ψ‐C‐G loop contains the most stable set of tertiary structure interactions.
Studies on the Mechanism of Action of Histone Kinase Dependent on Adenosine 3′:5′‐Monophosphate. Evidence for Involvement of Histidine and Lysine Residues in the Phosphotransferase Reaction
Authors: KOCHETKOV Sergei N. , BULARGCINA Tamara V. , SASHCHENKO Lidia P. , SEVERIN Evgenii S. 
Published: March 2005

The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3′: 5′‐monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping‐pong bi‐bi type.After incubation of the catalytic subunit of histone kinase with [γ‐32P]ATP the incorporation of one mole of [32P]phosphate per mole of protein was observed. The tryptic [32P]phosphohistidine‐containing peptide was isolated and its N‐terminus and amino acid composition were determined.The 2′,3′‐dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic‐AMP‐dependent histone kinase. The inhibitor formed an aldimine bond with ∈‐amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C‐labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP.Tryptic digestion of the enzyme‐inhibitor complex led to the isolation of the 14C‐labelled peptide of the active site of histone kinase.Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.
Röntgenkleinwinkel‐Untersuchungen und Strukturmodell‐Betrachtungen an der Fettsäuresynthetase aus Schweineleber
Authors: FOLKHARD Waltraud , FELSER Birgit , PILZ Ingrid , KRATKY Otto , DUTLER Hans , VOGEL Hugo 
Published: March 2005

Small‐Angle X‐Ray‐Scattering Investigation and Structural‐Model Study of the Fatty‐Acid Synthetase from Pig Liver The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetases from this and other sources. When comparing small‐angle X‐ray‐scattering curves calculated with these models and curves obtained from small‐angle X‐ray‐scattering experiments carried out with the pig‐liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an assymetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other.
Lipopolysaccharides de mutants thermosensibles de Escherichia coli K12
Authors: BRUNETEAU Maud , MICHEL Georges 
Published: March 2005

Lipopolysaccharides from Thermosensitive Mutants of Escherichia coli K12Thermosensitive mutants of Escherichia coli K12 were grown at 30°C and 40°C. The serologic properties and the composition of their lipopolysaccharides were investigated. The inhibition of hemagglutination by the lipopolysaccharides of various mutant strains was tested against the anti‐E. coli K12 CR34 system. An inhibition was observed with all the mutants but one, CR 34 T83 which had no inhibitory effect.Chemical analysis of lipopolysaccharides and mass spectrometric analysis of their methylated derivatives indicated the presence of the same components in the various lipopolysaccharides: glucose, galactopyranose, galactofuranose, heptopyranose and heptofuranose. However the 2,3,4 tri‐O‐methyl glucose is missing in the lipopolysaccharide of the mutant T83. This result agrees with the absence of a substituent on the 6‐position of the non‐reducing core‐terminal glucose. The lipopolysaccharide of the T83 mutant has the complete core‐type of E. coli K12. The relations of mutations with modifications of the composition of inner and outer envelopes in various mutants are discussed.
Colivirus‐T3‐Coded S‐Adenosylmethionine Hydrolase
Authors: SPOEREL Nikolaus , HERRLICH Peter 
Published: March 2005

Bacteriophage T3 induces an enzyme activity which hydrolyzes S‐adenosylmethionine. This S‐adenosylmethionine hydrolase is interesting, not only because of its unique activity, but also because the protein has to overcome host restriction [F. W. Studier and N. R. Movva (1976) J. Virol. 19, 136–145]. S‐Adenosylmethionine hydrolase was purified to homogeneity using affinity chromatography on S‐adenosylhomocysteine‐Sepharose. The enzyme occurs in two forms, A and B. Form A consists of the viral peptide chain only; its native and subunit molecular weight is 17000. Form B contains, in addition, a host subunit with a molecular weight of 49000. The host subunit does not modify S‐adenosylmethionine cleavage in vitro and no apparent relationship to the host‐restriction system could be detected.
Characterization of Pepsin Fragments of Basement Membrane Collagen Obtained from a Mouse Tumor
Authors: TIMPL Rupert , BRUCKNER Peter , FIETZEK Peter 
Published: March 2005

Basement membrane collagen was purified from an acetic acid extract of a mouse tumor and subjected to pepsin treatment. At initial stages disulfide‐linked molecules, consisting of peptide chains with an apparent molecular weight of 140000, were formed which were some 15–20% smaller than the chains in the original protein. In a second stage further fragmentation occurred producing at least five peptides ranging in molecular weight from 27000 to 72000. Each of these peptides was isolated and characterised by amino acid composition, cyanogen bromide cleavage, circular dichroism and antigenic activity.The data indicate that the major portion of the initial pepsin‐resistant fragment is degraded forming two triple‐helical segments, the disulfide‐linked fragment P3 and the cysteine‐free fragment P1 containing chains with molecular weights of 72000 and 55000 respectively. The other fragments (P2, P3B and P3C) were recovered at lower yields and may originate from a second, genetically distinct type of basement membrane collagen. This interpretation is supported by partial amino acid sequence analysis of the two non‐disulfide‐linked fragments (P1, P2). These sequences show that P1 and P2 are different and start with a non‐helical sequence followed by the helical triplet structure Gly‐X‐Y.Studies on pepsin fragments of the insoluble tumor collagen demonstrated a pattern similar to that obtained with the soluble form except for the occurrence of disulfide‐linked α‐chain‐like material. These chains also have an amino acid composition characteristic for basement membrane collagen. Taken together, the findings suggest that the tumor produces several related collagens.
Suppression of Thermotropic Lipid Clustering in Tetrahymena Nuclear Membranes upon Ca2+/Mg2+ Induced Membrane Contraction
Authors: GIESE Günter , FROMME Inge , WUNDERLICH Frank 
Published: March 2005

Nuclear membranes surrounding the macronuclei isolated from Tetrahymena cells grown at either 28 C or 18 C are induced to expand or contract laterally by Ca2+/Mg2+ (3/2) in final concentrations of 1 mM or 5 mM, respectively. Only expanded nuclear membranes are temperature‐responsive as revealed by freeze‐etch electron microscopy and electron spin resonance using 5‐doxylstearic acid as a spin label.In expanded nuclear membranes from growth at 28°C, electron spin resonance detects, upon lowering the temperature, a biphasic decrease of the lipid fluidity in terms of 2TII with a discontinuity at ∼ 17°C. In this temperature range, freeze‐etch electron microscopy reveals an increase in the number of nuclear membrane fracture faces with smooth areas. We ascribe this response to a clustering of ordered lipid domains within a broad thermotropic fluid→ordered lipid phase separation [Wunderlich et al. (1978) Biochemistry, 17, 2005–2010].Expanded nuclear membranes from growth at 18°C shift their lipid clustering temperature down to ∼ 12°C. Their lipid fluidity (2TII) is increased in comparison with the nuclear membranes from growth at 28°C. This is probably due to an increased fatty acid unsaturation of the polar lipids. Thin‐layer‐chromatography reveals the following major polar lipids in the nuclei from growth at 18°C (28°C): 33.9%(31.4%) phosphatidylethanolamine, 14.2%(13.4%) phosphatidylethylamine, 22.5%(18.0%) unidentified phospholipid, 21.4%(19.1%) phosphatidylcholine, and 6.1%(10.4%) ceramide‐phosphonoethylamine. The major fatty acids are mainly even‐numbered ranging between C12 and C18 as analyzed by gas chromatography. The 18°‐nuclei (28°‐nuclei) contain 67.1% (60.4%), 74.5%(68.4%), and 32.3%(43.5%) unsaturated fatty acids in their total lipids, polar lipids, and neutral lipids, respectively. The individual phospholipids, except phosphatidylcholine and ceramide‐phosphonoethylamine, are more unsaturated in the nuclei from growth at 18°C.Contracted nuclear membranes from growth at 28°C and 18°C suppress thermotropic lipid clustering. They reveal an apparent higher lipid fluidity and percentage of fracture faces with smooth areas than the corresponding expanded nuclear membranes at 28°C and 18°C, respectively. The lipid clustering suppression is interpreted as being due to a change in lipid packing which in turn is induced by a Ca2+/Mg2+‐induced contraction of the Ca2+/Mg2+‐sensitive nucleoskeleton proteins associated with nuclear membranes.
The Reaction of Nitrogen Monoxide and of Nitrite with Deoxyhaemocyanin and Methaemocyanin of Helix pomatia
Authors: VERPLAETSE Jozef , VAN TORNOUT Philippe , DEFREYN Ghislain , WITTERS Raphaël , LONTIE René 
Published: March 2005

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction.In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g= 4 and yielded a nitrosyl derivative with a characteristic g= 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra.Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g= 4 with 7 hyperfine lines.
Ribosomal Phosphoproteins of Mouse Myeloma Cells
Authors: MARTINI Oskar H. W. , KRUPPA Joachim 
Published: March 2005

Mouse myeloma cells (MPC 11) in culture, labelled with [32P]i for several hours, were subjected to hypertonic initiation block by raising the salt concentration of the culture medium from 110 to 210 mM NaCl.The relative amounts of messenger‐free and bound ribosomes of the cells were estimated from ribosome profiles following sucrose density centrifugation. The initiation block caused the proportion of messenger‐bound ribosomes to decrease substantially. The extent of the decrease was larger under suboptimal (50%) than under satisfactory (25%) nutritional conditions.Analysis of the ribosomal proteins by two‐dimensional gel electrophoresis, autoradiography and radiospectrometry showed, that in the small subunit only protein S3 had incorporated 32P and that S3 was the most strongly phosphorylated ribosomal protein in control cells. Upon salt treatment of the cells the radioactivity of S3 from both messenger‐free and bound ribosomes decreased to low levels. The lowest levels (15% of the controls) were reached when the nutritional conditions were suboptimal. The results show that there is a positive correlation between the number of ribosomes engaged in protein synthesis and the degree of phosphorylation of S3. However, phosphorylation of S3 does not seem to be a prerequisite for ribosomes to be attached to mRNA. Under all experimental conditions S3 from messenger‐bound ribosomes was about twice as strongly labelled as S3 from messenger‐free ribosomes.The large‐subunit phosphoproteins were identified as L28, the protein thought to be homologous to the Escherichia coli proteins L7/L12, and as L5, which was only weakly labelled. In contrast to S3 the radioactivity of L28 increased slightly upon the tonicity shift.
270‐MHz 1H Nuclear‐Magnetic‐Resonance Study of Met‐enkephalin in Water
Authors: ZETTA Lucia , CABASSI Franco , TOMATIS Roberto , GUARNERI Mario 
Published: March 2005

1H spectra at 270 MHz of the Met‐enkephalin pentapeptide (Tyr‐Gly‐Gly‐Phe‐Met) in aqueous solution, as a function of pH and of temperature, are reported.The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around χ1 and χ2, suggests the existence of three conformational regions in the pH range 1–10, due to some rearrangements of the first two amino acids, Tyr‐1 and Gly‐2. A first conformational transition occurs corresponding to the N‐terminus titration, with an exchange rate between conformational states which is not too fast. The second transition occurs concomitantly with the phenolic group titration.The conformational states of the cationic and zwitterionic forms are probably very similar and are characterized by the absence of intramolecular hydrogen bonds and of head‐to‐tail interactions. A bent structure, with the Tyr‐1 ring Facing the Gly‐2 could contribute to the equilibrium of the conformational states of Met‐enkephalin in the pH range 8–10. This structure could be destabilized as a consequence of the phenolic group deprotonation.The Met‐5 and Phe‐4 residues appear to have little sensitivity to the conformational transitions of the backbone and both show a settling of the side chains corresponding to the carboxyl group titration, thus showing a reciprocal steric hindrance.The temperature coefficients of the chemical shifts of amide protons at acidic pH are in the range of solvated peptides. In the temperature range 0–75°C, the 3JαNH coupling constants were independent of temperature.
Characterization of the Isoenzymes of Pig‐Liver Esterase 2. Kinetic Studies
Authors: JUNGE Wolfgang , HEYMANN Eberhard 
Published: March 2005

The kinetic properties of two of the partially separated isoenzymes I and V of pig liver esterase were studied. The cholinesterase‐like isoenzyme I hydrolyses butyrylcholine as well as various other esters and aromatic amides. This isoenzyme is sensitive to 0.01 mM physostigmine and to fluoride. The second type (isoenzyme V) has the features of the so‐called aliesterase: it acts preferentially on short‐chain aliphatic esters, does not hydrolyse butyrylcholine and has only low activity towards amides. Further differences exist with regard to sensitivity towards organophosphorous inhibitors, to the influence of organic solvents and to the pH optimum. Transacylation reactions with methanol as an acceptor are mainly catalyzed by the isoenzyme of the aliesterase type.The esterase forms III and IV, which are located between isoenzymes I and V on the isoelectric focussing column, show kinetic features similar to those of a mixture of I and V. A stepwise increase or decrease, respectively, of certain kinetic properties, e.g. specific activities, is observed for the sequence I, III, IV, V. A quantitative comparison of the kinetic properties supports our proposed subunit model [1], according to which the main components of these four esterase fractions are the trimers γγγ, αγγ, ααγ and ααα.These results offer explanations for many of the very complex and often controversial results formerly obtained with the heterogeneous pig liver esterase.
The Molecular Size and Shape of the Folch‐Pi Apoprotein in Aqueous and Organic Solvents
Authors: LAVIALLE Françoise , de FORESTA Béatrice , VACHER Monique , NICOT Claude , ALFSEN Annette 
Published: March 2005

In 1% acetic acid, sedimentation velocity measurements and equilibrium ultracentrifuge experiments demonstrate that the Folch‐Pi apoprotein is not monodisperse. The weight‐average molecular weight calculated from ultracentrifuge experiments and combining sedimentation coefficient and viscosity measurements, ranged from 64000 to 80000. The intrinsic viscosity value suggests an asymetric shape for the apoprotein if a low value of hydration is considered.In dioxan/1% acetic acid (2:3, v/v) a smaller sedimentation coefficient was found, the intrinsic viscosity value remaining identical to that in 1% acetic acid.In pure 2‐chloroethanol, light‐scattering experiments led to a molecular weight of 165000 indicating that even in this solvent the protein is not monomeric. Intrinsic viscosity and light scattering measurements on the one hand, primary sequence on the other hand (six proline residues per monomer of Mr 23500) suggest that the molecule in 2‐chloroethanol may consist of rod‐like segments with flexible junctions.
Inactivation and Disassembly of the Pyruvate Dehydrogenase Multienzyme Complex from Bovine Kidney by Limited Proteolysis with an Enzyme from Rat Liver
Authors: KRESZE Georg‐B. , STEBER Liesel 
Published: March 2005

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin‐sensitive enzyme (termed ‘inactivase’) obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex.By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high‐molecular‐weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.
Optical Probe Responses on Sarcoplasmic Reticulum Oxacarbocyanines as Probes of Membrane Potential
Authors: BEELER Troy , RUSSELL James T. , MARTONOSI Anthony 
Published: March 2005

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on saracoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential.3.3′‐Diethyloxodicarbocyanine responds to ATP‐induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump‐mediated Ca2 fluxes.A large change in the absorbance of 3,3′‐diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient.The maximum of the potential‐dependent absorbance change is at 575–580 nm. The potentials generated by valinomycin or ionophore A23187 are short‐lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.
Methionyl‐tRNAfMet Deacylase
Authors: ANDERSEN Klaus B. 
Published: March 2005

A methionyl‐tRNAfMet deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE‐cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80000, which consists of two 40000‐Mr subunits.The mechanism of the Met‐tRNAfMet deacylase is shown to involve end‐product inhibition by the deacylated form of MettRNAfMet. The methionyl‐tRNAfMet deacylase is rather specific for Met‐tRNAfMet as opposed to Met‐tRNAmMet, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40‐S and 60‐S subunits inhibit the enzyme, possibly by binding to it.The stability of translational initiation complexes, containing methionyl‐tRNAfMet, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40‐S‐subunit · Met‐tRNAfMet complex was stable in the presence of enzyme. The 80‐S complex formed with A‐U‐G trinucleotide as the message molecule was broken down, whereas the 80‐S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met‐tRNAfMet.
Cobra Venom Acetylcholinesterase
Authors: RABA Raivo , AAVIKSAAR Aavo , RABA Mari , SIIGUR Jüri 
Published: March 2005

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate.Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67000 ± 2000. At high enzyme concentrations (> 0.2 mg/ml, > 1 μM) and ionic strength 0.1 M, it reversibly tends to form higher‐molecular‐weight 7.1‐S aggregates.Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.