bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell‐Wall Lipopolysaccharide of the ‘Shigella‐like’Escherichia coli 058
Authors: DMITRIEV Boris A. , KNIREL Yuriy A. , KOCHETKOV Nikolay K. , JANN Barbara , JANN Klaus 
Published: March 2005

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical anylysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O‐specific polysaccharide moiety.The O‐specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas‐liquid chromatography and ion‐exchange chromatography, it was shown to contain D‐mannose, 2‐acetamido‐2‐deoxy‐D‐glucose, 3‐O‐(‐R‐1′‐carboxyethyl)‐L‐rhamnose (rhamnolactylic acid). and O‐acetyl groups in the molar ratios of 2: 1: 1: 1.The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide β‐GlcNAcl – 4αMan‐1–4(2/3‐O‐Ac)‐Man is substituted at C‐3 of the non‐acetylated mannose with rhamnolactylic acid. The repeating units are joined through α‐mannosyl‐1–3‐glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5.
Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei
Authors: BLANCHARD Jean‐Marie , BRUNEL Claude , JEANTEUR Philippe 
Published: March 2005

Ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) were isolated from sonically disrupted HeLa cells nuclei by sedimentation through a triple sucrose cushion. We found that these particles were able to catalyse the incorporation of radioactive phosphate from [γ32P]ATP into phosphoserine and phosphothreonine residues of endogenous proteins. Optimal conditions for enzymatic activity were defined after investigation of the influence of several parameters of the reaction. The requirement for a divalent cation was most efficiently met by 10 mM Mg2+, to a lower extent by Mn2+. An apparent Km value for ATP of 0.02 mM was determined and the enzyme was found to be sensitive to p‐hydroxymercuribenzoate. Neither cyclic AMP nor cyclic GMP stimulated the reaction over a wide range of concentrations and the same protein substrates were phosphorylated in their presence.After excluding possible contamination by other subcellular fractions, considerable evidence for the association of kinase and phosphate acceptor proteins with ribonucleoprotein structures containing hnRNA was gathered along several lines by showing that the enzymatic activity closely followed tritiated RNA pulse‐labeled in the presence of low doses of actinomycin D in the following circumstances: (a) sedimentation in sucrose gradients, (b) isopycnic banding in metrizamide density gradients in the presence or absence of Mg2+ ions and (c) adsorption to oligo(dT)‐cellulose columns under conditions where poly(A)‐containing material is adsorbed.Analysis of phosphorylated substrates by electrophoresis on polyacrylamide gels containing sodium dodecylsulphate revealed that, although they spanned a wide range of molecular weights, the most intensely labeled species were localized in bands corresponding to molecular weights of 37000 and 28000.The above pattern of polypeptides phosphorylated in vitro is compared with that of phosphoproteins labeled in vivo after exposure of cells to [32P]phosphate.
Reaction of Yeast Fatty Acid Synthetase with Iodoacetamide
Authors: OESTERHELT Dieter , BAUER Heidi , KRESZE Georg‐Burkhard , STEBER Liesel , LYNEN Feodor 
Published: March 2005

Yeast fatty acid synthetase is inactivated by several SH reagents. For the irreversible inhibition by iodoacetamide, the apparent second‐order rate constant k= 1.3 ± 0.2 M−1 s−1 was found (0 °C, pH 6.5). This value is about 125‐times higher than the rate constant for the reaction between iodoacetamide and free cysteine under the same conditions and was found to be pH‐independent in the range between pH 5 and pH 9. A possible explanation for this result might be that the SH‐group alkylated by iodoacetamide is hydrogen‐bonded to a neighbouring basic group in the protein.Of all partial activities of the synthetase, only the condensation reaction is impaired. The enzyme can be protected against iodoacetamide by prior treatment with acetyl‐CoA but not malonyl‐CoA. This indicates that iodoacetamide reacts with peripheral SH‐groups of the multienzyme complex. N‐Ethylmaleimide, in contrast, inhibits the synthetase in a strongly pH‐dependent manner. These kinetic data support the conclusion drawn from earlier studies on the malonyl binding sites of the enzyme [Schweizer, E., Piccinini, F., Duba, C., Günther, S., Ritter, E. and Lynen, F. (1970) Eur. J. Biochem. 15, 483–499] that the reaction of N‐ethylmaleimide with another SH‐group of the enzyme is responsible for the inactivation. During the reaction with iodoacetamide, fatty acid synthetase activity is completely destroyed after incorporation of about three carbamoylmethyl residues per molecule of enzyme complex. At 0 °C, further alkylation by iodoacetamide could not be detected. Since yeast fatty acid synthetase is proposed to be an A6B6 hexamer, the possibility of half‐site reactivity is considered.
Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer
Authors: ZELIKSON Ruth , LUZZATI Mario 
Published: March 2005

The study of the subcellular distribution of the enzymes involved in folate‐coenzyme‐mediated transfer of one‐carbon groups in Saccharomyces cerevisiae suggests the existence of two parallel enzyme systems, one mitochondrial and the other cytoplasmic. All the enzymes assayed in the present study, serine transhydroxymethylase, dihydrofolate reductase, 5,10‐methylenetetrahydrofolate dehydrogenase, 10‐formyltetrahydrofolate synthetase, 5,10‐methylenetetrahydrofolate reductase and thymidylate synthetase were found to be in both cytoplasm and mitochondria. Two of the mitochondrial enzymes, serine transhydroxymethylase and dihydrofolate reductase, were found to be different by several criteria from their cytoplasmic counterparts.By the use of three nuclear gene mutations ade3, tmp3 and serl, and different combinations of these three mutations, the physiological role of the cytoplasmic enzymes for the biosynthesis of adenine, histidine, methionine, thymidylate and serine was studied. The response of auxotrophes to an external source of tetrahydrofolate, i.e. 5‐formyl tetrahydrofolic acid, was also examined. The mitochondrial set of enzymes might be responsible for the de novo formation of tetrahydrofolate, since the loss of the three mitochondrial enzyme activities, serine transhydroxymethylase, thymidylate synthetase and dihydrofolate reductase, due to the mutation tmp3, leads to a generalized requirement for thymidylate, methionine, adenine and histidine. The ade3 mutation in which the cytoplasmic activities of the 10‐formyltetrahydrofolate synthetase, 5,10‐methenyltetrahydrofolate cyclohydrolase and 5,10‐methylenetetrahydrofolate dehydrogenase are lost, confers only adenine and histidine auxotrophy, showing that the ade3 proteins are synthesized in, and only function in, the cytoplasm. The mitochondrial counterparts of these enzymes may function solely in the formation of the 10‐formyl tetrahydrofolate needed for the transformylation of fMet‐tRNA involved in mitochondrial protein synthesis.
Investigation of the Binding of Escherichia coli RNA Polymerase to DNA from Bacteriophages T2 and T7 by Kinetic Formaldehyde Method and Electron Microscopy
Authors: CHERNY Dimitrii I. , ALEKSANDROV Aleksander A. , ZARUDNAYA Margarita I. , KOSAGANOV Yurii N. , LAZURKIN Yurii S. , BEABEALASHVILLI Robert Sch. , SAVOCHKINA Larisa P. 
Published: March 2005

The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy. For electron‐microscopic investigations the effect of different conditions of formaldehyde fixation for DNA · RNA‐polymerase complexes was studied and optimal fixation conditions were found. The suggested fixation method for DNA · RNA‐polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ratio of enzyme to DNA etc.).The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence. The electron‐microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA. A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found.
ATPase Activities, Ca2+ Transport and Phosphoprotein Formation in Sarcoplasmic Reticulum Subfractions of Fast and Slow Rabbit Muscles
Authors: HEILMANN Claus , BRDICZKA Dieter , NICKEL Elvira , PETTE Dirk 
Published: March 2005

Subfractionation of sarcoplasmic reticulum from fast‐twitch and slow‐twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca2+,Mg2+)‐dependent (extra) ATPase, Mg2+ ‐dependent (basal) ATPase, Ca2+ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electro‐phoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples.In fast‐twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca2+, Mg2+)‐dependent ATPase, negligible activity of Mg2+‐dependent ATPase, high initial rate and high capacity of Ca2+ uptake, high amount of phosphorylated 115000‐Mr polypeptide, and appeared morphologically as thin‐walled vesicles covered with particles of 4 nm in diameter.Low density vesicles had little (Ca2+, Mg2+)‐dependent ATPase but high Mg2+‐dependent ATPase. Although the initial rate of Ca2+ uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000‐Mr polypeptide was detectable at low concentrations. Instead, 57000 and 47000‐Mr polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg2+. Negatively stained, these vesicles appeared to have smooth surfaces.It is suggested that low density vesicles represent a Ca2+ sequestering system different from that of high density vesiclés and that Mg2+‐dependent (basal) ATPase as well as the 57000 and 47000‐Mr polypeptides are part of the Ca2+ transport system within the low density vesicles. According to the results from slow‐twitch muscle, Ca2+ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the low density vesicles.
Temperature‐Sensitive Mutants of Escherichia coli with Defects in the Assembly of RNA Polymerase in vitro
Authors: GROSS Gerhard C. , FIELDS Dorothy A. , BAUTZ Ekkehard K. F. 
Published: March 2005

RNA polymerase was purified from three temperature‐sensitive mutants of Escherichia coli, one (A2R7) mapping in rpoB, the second (R120) in rpoC, and the third (R60‐39), which has not been genetically mapped as yet, but which was found to produce a temperature‐sensitive β subunit. The enzymes were tested for their ability to reconstitute active enzyme from isolated subunits. RNA polymerase from A2R7 showed a reduced level of reconstitution compared to wild‐type enzyme at 30°C while no reconstitution was observed if the subunits were incubated at 42°C, the non‐permissive temperature for growth of the mutant bacteria. Wild‐type enzyme shows nearly full reconstitution at this temperature. Once assembled, the A2R7 holoenzyme complex is temperature resistant. The temperature‐sensitive lesion could be assigned to the β subunit, and it appears that the assembly is blocked primarily at a step involving the addition of the β′ subunit.The RNA polymerase of R120 fails to reconstitute to an active holoenzyme at any temperature. Mixed reconstitution experiments with wild‐type subunits showed that the subunit responsible for this lack of reconstitution is β′. The holoenzyme itself is temperature resistant.The RNA polymerase of R60‐39 assembles normally in vitro at 35°C but fails to assemble at 42°C. The temperature‐sensitive lesion of the RNA polymerase is due to its β′ subunit and, as in the case of A2R7, the assembly seems to be blocked at a step involving the addition of β′ to the α2β complex at 42°C. The holoenzyme itself shows only a slightly reduced rate of RNA synthesis at 42°C.We conclude that for all three mutants the temperature‐sensitive lesions in vivo are due to a failure of the newly synthesized polypeptide chains to form an active holoenzyme complex at the non‐permissive temperature.
A Calcium‐Dependent Protein Activator of Guanosine 3′:5′‐Monophosphate Phosphodiesterase in Bovine and Rat Pancreas
Authors: VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , RATHÉ Jean , KUTZNER Rodolphe , DELFORGE Alain , CHRISTOPHE Jean 
Published: March 2005

A protein activator for guanosine 3′:5′‐monophosphate phosphodiesterase from bovine pancreas was purified in high yield and to apparent homogeneity as judged by standard polyacrylamide gel and sodium dodecylsulfate polyacrylamide gel electrophoresis. The purification required heat treatment, ammonium sulfate precipitation, ion‐exchange chromatography on DEAE‐cellulose and gel filtration on Sephadex G‐75.The minimum molecular weight of the activator calculated from the amino acid composition was 18779. Acid hydrolysates contained a high content of aspartic and glutamic acids, a high phenylalanine to tyrosine ratio, and one ɛ‐N‐trimethyllysine per mole protein. No cysteine and tryptophan were detected in the protein.The soluble supernatant fraction of rat pancreas homogenate subjected to gel filtration on Sephadex G‐200 revealed 3 peaks (P1, P2 and P3) with cyclic nucleotide phosphodiesterase activity and one peak of the protein activator. P1, P2 and P3 phosphodiesterases were all active to varying extent on both cyclic GMP and cyclic AMP. The activity of P3 with cyclic GMP as substrate was specifically stimulated three–five‐fold by the purified bovine pancreatic protein activator in the presence of free Ca2+. Phosphodiesterases P1 and P2 were not stimulated by this activator and were not inhibited by the Ca2+‐chelating agent, EGTA.A comparison of the phosphodiesterase activity of a crude rat pancreas extract in the presence of Ca2+ or EGTA, showed that 42% of low‐Km cyclic GMP phosphodiesterase activity was responsive to the Ca2+‐activator complex while cyclic AMP phosphodiesterase was not. In addition, a large excess of protein activator appeared to exist in the rat pancreas.
Studies on DNA Unwinding
Authors: GARSSEN Gerrit J. , HILBERS Cornelis W. , SCHOENMAKERS John G. G. , van BOOM Jacques H. 
Published: March 2005

The interaction of gene V protein from bacteriophage M13 with the self‐complementary tetra‐nucleotide d(pC‐G‐C‐G) was studied by 1H and 31P nuclear magnetic resonance. It is shown, using the hydrogen‐bonded proton resonances of the Watson‐Crick base pairs as a probe, that the protein is able to unwind the small double‐helical fragment even at 0 °C.Binding of the tetranucleotide causes changes in the aromatic part of the 1H NMR spectrum of the complex, suggesting that aromatic residues, most likely tyrosines, take part in the protein · nucleic‐acid interaction. From the 31P NMR spectra of the protein · nucleic‐acid complex it follows that the pK value of the 5′‐terminal phosphate is lower than for the free nucleic acid species. Moreover, it could be shown that the exchange of the protein between nucleic acid substrates is fast.Combination of these measurements has led us to derive a mechanism of unwinding on the tetranucleotide level. To a large extent the unwinding is determined by fluctuations in the double‐helical DNA structure.
Mechanism of Chymotrypsin: Acid/Base Catalysis and Transition‐State Solvation by the Active Site
Authors: FASTREZ Jacques , HOUYET Nicole 
Published: March 2005

An acyl‐enzyme of a modified chymotrypsin, in which the Nɛ2 nitrogen of histidine‐57 is methylated, has been prepared. The rates of deacylation of this modified acyl‐enzyme have been determined as a function of the pH at 25°C. The pH profile reveals two concurrent pathways of deacylation: below pH 8.0 the deacylation follows a titration curve similar to that found with the native enzyme with a pKa close to 7.0 but a maximal rate constant 4 × 104 times lower; above pH 9.0 a hydroxide‐ion‐catalyzed pathway becomes significant.The deuterium isotope effect and the thermodynamic parameters ΔH≠ and ΔS≠ have been determined for both pathways; the results are consistent with the interpretation of the low‐pH pathway as a general base catalysis by a residue of the active site and the high‐pH pathway as the basic hydrolysis of the ester linkage within the active site. There are, nevertheless, small differences between the values expected for these parameters for a reaction in solution and within the active site of the enzyme.The rates of deacylation are also increased by high concentrations of trimethylamine or imidazole buffers; this observation is tentatively interpreted as a general base catalysis by tertiary amines. Comparison of these data with literature results on the rates of deacylation of the native enzyme and hydrolysis of esters in solution leads to the conclusion that the efficiency of acid–base catalysis is more important than tetrahedral intermediate solvation by the active site of the enzyme as a contribution to chymotrypsin catalysis.
Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in a Cell Free System from Neurospora crassa
Authors: HARMEY Matthew A. , HALLERMAYER Gerhard , KORB Harald , NEUPERT Walter 
Published: March 2005

Synthesis and transport of mitochondrial proteins were followed in a cell‐free homogenate of Neurospora crassa in which mitochondrial translation was inhibited. Proteins synthesized on cytoplasmic ribosomes are transferred into the mitochondrial fraction. The relative amounts of proteins which are transferred in vitro are comparable to those transferred in whole cells.Cycloheximide and puromycin inhibit the synthesis of mitochondrial proteins but not their transfer into mitochondria.The transfer of immunoprecipitable mitochondrial proteins was demonstrated for matrix proteins, carboxyatractyloside‐binding protein and cytochrome c.Import of proteins into mitochondria exhibits a degree of specificity. The transport mechanism differentiates between newly synthesized proteins and preexistent mitochondrial proteins, at least in the case of matrix proteins.In the cell‐free homogenate membrane‐bound ribosomes are more active in the synthesis of mitochondrial proteins than are free ribosomes. The finished translation products appear to be released from the membrane‐bound ribosomes into the cytosol rather than into the membrane vesicles.The results suggest that the transport of cytoplasmically synthesized mitochondrial proteins is essentially independent of cytoplasmic translation; that cytoplasmically synthesized mitochondrial proteins exist in an extramitochondrial pool prior to import; that the site of this pool is the cytosol for at least some of the mitochondrial proteins; and that the precursors in the extramitochondrial pool differ in structure or conformation from the functional proteins in the mitochondria.
Human Platelet Actin
Authors: LANDON Françoise , HUC Claude , THOMÉ Françoise , ORIOL Christine , OLOMUCKI Anna 
Published: March 2005

Human blood platelet actin was purified using 30% sucrose to extract actomyosin and potassium iodide to dissociate actomyosin and to depolymerize actin.Pure actin thus obtained resembles skeletic muscle actin in its polymerization properties, CD spectra and ability to activate myosin Mg2+‐ATPase.Isoelectric focusing gel analysis shows that human blood platelet actin exists in β and γ forms. The ratio of β to γ forms is of 5 in purified actin, in whole cell extract and in all the fractions studied.
Covalent Structure of Collagen: Amino‐Acid Sequence of Chymotryptic Peptides from the Carboxyl‐Terminal Region of α2‐CB3 of Chick‐Skin Collagen
Authors: DIXIT Saryu N. , SEYER Jerome M. , KANG Andrew H. 
Published: March 2005

The amino acid sequence of chymotryptic peptides C4 and C5 which together make up 206 COOH‐terminal residues of α2‐CB3 of chick skin collagen is described. This in combination with the sequence of 132 residues from the amino‐terminal region published earlier [Dixit, Seyer, and Kang (1977) Eur. J. Biochem. 73, 213–221] completes the total amino acid sequence of the large CNBr peptide, α2‐CB3 of chick skin collagen.The amino acid sequence was determined by automated Edman degradation of intact peptides C4 and C5 and their respective tryptic and maleylated tryptic peptides, and thermolytic peptides of C4. The comparison of the sequence with the homologous segment of α1(I) chain showed striking variance of over 51% within the same species.
Production of Antibodies against ADP‐ribose and 5′‐AMP with the Aid of N6‐Carboxymethylated ADP‐ribose Conjugates
Authors: BREDEHORST Reinhard , FERRO Ari M. , HILZ Helmuth 
Published: March 2005

Adenosine diphosphate ribose (ADP‐ribose) coupled directly to bovine serum albumin by the carbodiimide method resulted in a low production of antibodies with a preferential affinity for 5′‐AMP. To allow condensation of the ADP‐ribose residue in a more specific manner, conjugates of N6‐carboxymethylated ADP‐ribose (cm6ADP‐ribose) and serum albumin were synthesized in two ways. First, adenine‐N6‐carboxymethylated NAD (cm6NAD) was coupled by carbodiimide to serum albumin nearly exclusively through the carboxyl group as judged from a comparison of the epitope density (number of haptens/albumin) of the conjugate with an antigen prepared from unmodified NAD. However, only incomplete enzymic transformation of the NAD hapten to an ADP‐ribose hapten could be achieved due presumably to steric hindrance.Second, cm6ADP‐ribose was synthesized from cm6NAD by treatment with NAD glycohydrolase. This novel compound was coupled to serum albumin or to methylated serum albumin with the aid of carbodiimide. Only conjugates obtained with the last‐mentioned protein acceptor elicited significant titers of ADP‐ribose antibodies besides a similarly high production of 5′‐AMP antibodies.With cm6ADP‐ribose coupled to non‐methylated serum albumin, 5′‐AMP antibodies were produced exclusively, which showed an excellent discrimination between 5′‐AMP and 3′‐AMP, or AMP residues in nucleic acids. Production of 5′‐AMP antibodies was presumably caused by hydrolytic breakdown of the ADP‐ribose antigen in the recipient animals.