bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleolar Chromatin in Chinese Hamster Ovary Cells
Authors: BACHELLERIE Jean‐Pierre , NICOLOSO Monique , ZALTA Jean‐Pierre
Published: March 2005

A stepwise physico‐chemical dissection of nucleoli isolated from Chinese hamster ovary cells has allowed the isolation of actively transcribed regions of chromatin, highly enriched in ribosomal genes. Nucleolar chromatin could be separated into its various topographical components. This separation should improve our knowledge of the structure/function relationships in chromatin as the fine structure of the nucleolus is very sensitive to changes in ribosomal RNA synthesis.

The spatial distribution of ribosomal genes among the various chromatin areas of the nucleolus was assayed by hybridization with rRNA. In exponentially growing Chinese hamster ovary cells ribosomal genes are predominantly located in the intranucleolar stretches of nucleolus‐associated chromatin. However, ribosomal sequences represent but a very minor part (less than 1 %) of intranucleolar DNA. In an attempt to improve the analysis of the actively transcribed chromatin areas, intranucleolar chromatin was submitted to additional fractionation steps involving, among others, isopycnic separation in metrizamide gradients. In these latter conditions a small subset of intranucleolar chromatin banded with preribosomal ribonucleoprotein. In this subfraction, about one‐third of the DNA sequences corresponded to preribosomal RNA matrix units. The yield of recovery of this intranucleolar chromatin subfraction being closely related to the level of transcriptional activity of ribosomal genes, it appears likely that this separating procedure depends on the presence of ribosomal RNA transcription complexes and could, therefore, represent a general procedure for the isolation of actively transcribed areas from chromatin.
DNA Unwinding Enzyme II of Escherichia coli
Authors: ABDEL‐MONEM Mahmoud , CHANAL Marie‐Christine , HOFFMANN‐BERLING Hartmut
Published: March 2005

A DNA‐stimulated ATP‐γ‐phosphohydrolase of molecular weight 75000 was purified from Escherichia coli cells. The ATPase, a globular molecule (identical probably with an ATPase described previously by Richet and Kohiyama in 1976) shows specificity for adenine nucleotides, it prefers single‐stranded DNA as the cofactor, it exhibits a complicated mode of response to variations of the cofactor concentration and it is devoid of nuclease activity. Preparations derived from rep3 mutant cells yield widely varying amounts of an apparently normal ATPase.
The Reversible Inhibition of Gluconeogenesis in Kidney Cortex by Diazenedicarboxylic Acid Bis(N,N‐dimethylamide)
Authors: PILLION Dennis , LEIBACH Frederick H. , ROCHA Hector , VON TERSCH Francis J. , MENDICINO Joseph
Published: March 2005

Gluconeogenesis in rat kidney cortex slices was reversibly inhibited by diazenedicarboxylic acid bis(N,N‐dimethylamide). The rate of formation of glucose from pyruvate, malate, succinate and D‐fructose decreased at least 3‐fold in the presence of this inhibitor, commonly called diamide. The extent of inhibition of gluconeogenesis from pyruvate was dependent on the concentration of diamide and closely correlated with the degree of inhibition of protein kinase. The inhibition of gluconeogenesis and protein kinase were completely reversed after removal of the inhibitor. The effects of diamide on particulate enzymes which may be involved at rate‐limiting steps in transport processes which support gluconeogenesis in renal tissue was examined. Several enzymes present in kidney brush border and microsomal membrane preparations were inhibited by diamide. Na+, K+‐dependent ATPase and glucose‐6‐phosphatase were inhibited, but Mg2+‐dependent ATPase, alkaline phos‐phatase and y‐glutamyl transpeptidase, which are also present in these same subcellular fractions, were not inhibited by diamide. Based on the results obtained in these studies it is proposed that diamide may inhibit gluconeogenesis at rate‐limiting steps catalyzed by enzymes which are regulated by protein kinase. These enzymes, in turn, appear to play a role in the transfer of substrates across the plasma membrane.

The mechanism of inhibition of protein kinase by diamide was also investigated. Protein kinase present in a wide variety of rat tissues as well as phosphorylase kinase and pyruvate dehydrogenase kinase was inhibited by diamide. Kinetic studies showed that the inhibition of protein kinase by diamide was non‐competitive with respect to Mg2+, ATP, protein substrates and cyclic AMP. Sedimentation studies in sucrose‐density gradients containing histone and cyclic AMP showed that diamide inhibited both the intact and dissociated forms of protein kinase. These results support earlier observations which showed that diamide did not bind at the active site of protein kinase, but instead formed inactive complexes by binding to hydrophobic areas on the surface of the enzyme.
Further Evidence that Elongation Factor 1 Remains Bound to Ribosomes during Peptide Chain Elongation
Authors: GRASMUK Hans , NOLAN Robert Dwyer , DREWS Jürgen
Published: March 2005

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF‐1) and elongation factor 2 (EF‐2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti‐L7/L12 proteins on the binding and function of the two factors. It is further shown that EF‐1/β from Artemia salina does not function in displacing EF‐1 from mouse ascites tumor cell ribosomes.

These results also support our recently proposed model that EF‐1 remains bound to the ribosome during the peptide chain elongation cycle.
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.
Polypeptide Chains of 19‐S Thyroglobulin from Several Mammalian Species and of Porcine 27‐S Iodoprotein
Authors: MARRIQ Claudine , ROLLAND Marcel , LISSITZKY Serge
Published: March 2005

Undegraded 19‐S thyroglobulin was purified from hog, ox, man, dog, sheep and rat thyroid glands. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the reduced proteins showed that all are formed of peptide chains of molecular weight about 330000. Carboxypeptidase A digestion of porcine 19‐S thyroglobulin released consecutively two moles of leucine and then two moles of serine, thus offering strong evidence in favour of the idea that the protein is formed of two identical chains. The same C‐terminal amino acids were detected in sheep, ox, dog and man thyroglobulins. No N‐terminal amino acid was found by appropriate chemical and enzymatic techniques. Porcine 27‐S iodoprotein was shown by carboxypeptidase A analysis to be formed of four single‐stranded 330000‐Mr subunits identical to those constituting the 19‐S protein. Identity, both qualitatively and quantitatively, of the peptides obtained by CNBr cleavage of the two proteins as shown by sodium dodecylsulfate/polyacrylamide gel electrophoresis has confirmed this conclusion. Since 19‐S and 27‐S thyroid iodoproteins are formed of two and four probably identical chains, they must be termed 19‐S and 27‐S thyroglobulins or alternatively thyroglobulin dimer and tetramer, respectively.
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.
A Small‐Angle X‐Ray Investigation of the Satellite Tobacco Necrosis Virus
Authors: SJÖBERG Bo
Published: March 2005

Small‐angle X‐ray data recorded from solutions of satellite tobacco necrosis (STN) virus, in the concentration range 3.7–15.0 mg/cm3 are presented. The data were analysed using a least‐squares computer program where the experimental, slit‐smeared intensity function is used directly, without desmearing. The radius of gyration of the virus particle is 6.80 nm. From a scale factor obtained from the least‐squares analysis, and the isopotential specific volume, which was determined to 0.683 cm3/g, the total molecular weight of the monomeric STN virus was determined to 1.70 × 106. In addition to the monomers, all the solutions investigated contained about 1% (by weight) of the virus in the form of polymers, most probably aggregates of 12 monomers. From the chemical composition and the total molecular weight of the STN virus the molecular weight of the RNA was found to be 0.34 × 106 and the total molecular weight of the protein to be 1.36 × 106. With 60 protein subunits/virus particle the protein subunit molecular weight is 22700. In order to obtain some information about the internal structure of the virus, a model built of concentric spherical shells of equal thickness was constructed; and the average excess electron density of each shell, as well as the radius of the virus particle, was determined with the least‐squares computer program. At least two different electron density profiles can explain the experimental data. One of these has a rather low electron density at the centre of the particle and is, therefore, on the basis of other available information, considered as the most probable one. From the electron density distribution thus obtained a model of the virus particles was constructed in the following way. It was first assumed that the RNA forms a central core which is surrounded by the protein and that there is no penetration of the RNA into the protein domain, or vice versa. Then the water content of each shell was calculated by packing RNA, protein and water into the different shells in the proportions required so that the appropriate electron densities were obtained. As a result of these calculations it follows that the main part of the RNA is contained within the region 1.4–5.6 nm and the majority of the protein in the region 5.6–8.4 nm from the centre of the particle.
Joining of Synthetic Ribotrinucleotides with Defined Sequences Catalyzed by T4 RNA Ligase
Authors: OHTSUKA Eiko , NISHIKAWA Satoshi , FUKUMOTO Ryoichi , TANAKA Shoji , MARKHAM Alexander Fred , IKEHARA Morio , SUGIURA Masahiro
Published: March 2005

RNA ligase catalyzed the joining of pC‐C‐Ap with C‐A‐A in the synthesis of C‐A‐A‐C‐C‐Ap, which has the sequence of the Escherichia coli tRNAMetf 3′‐end. pC‐C‐A was also shown to be joined to C‐A‐A without any undesired self‐polymerization. Joining of pC‐C‐A to various synthetic ribotriplets, such as C‐C‐A, A‐A‐A, C‐C‐C, U‐U‐U, U‐A‐G, C‐C‐G and U‐U‐C, was performed as well as joining to the partially substituted trimers with a photolabile o‐nitrobenzyl group, C‐Anbzl‐A and C‐C‐Anbzl. The yields were C‐A‐A‐C‐C‐A (69%), C‐C‐A‐C‐C‐A (38%), A‐A‐A‐C‐C‐A (66%), C‐C‐C‐C‐C‐A (71%), U‐U‐U‐C‐C‐A (50%), U‐A‐G‐C‐C‐A (23%), C‐C‐G‐C‐C‐A (43%) and U‐U‐C‐C‐C‐A (46%). C‐Anbzl‐A was a slightly poorer acceptor than C‐A‐A and C‐C‐Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme.
Changes in Enzyme Activities during the Artificially Stimulated Transition from Follicular to Luteal Cell Types in Rat Ovary
Authors: KLINKEN Svend P. , STEVENSON Patricia M.
Published: March 2005

This paper reports the time course of development of the intramitochondrial cholesterol side‐chain‐cleavage activity, cytoplasmic NADP+‐dependent isocitric dehydrogenase, malic enzyme and glucose‐6‐phosphate dehydrogenase during ovarian maturation, using as a model the immature rat ovary stimulated to develop with pharmacological doses of gonadotrophin. These enzymic activities were correlated with increases in ovarian content of DNA, cellular content of adenosine 3′:5′‐monophosphate, and the levels of plasma progesterone. The plasma progesterone concentrations followed closely the development of the [4‐14C]cholesterol side‐chain‐cleavage which was mimicked by the cytoplasmic isocitric dehydrogenase; both enzymes increased in activity 28 times during the 6 days of this study. There was no correlation between adenosine 3′:5′‐monophosphate levels and cholesterol side‐chain cleavage or progesterone plasma concentrations.
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.
Adenylylation/Deadenylylation Control of the Glutamine Synthetase of Rhodopseudomonas capsulata
Authors: JOHANSSON Bo C. , GEST Howard
Published: March 2005

Previous attempts to demonstrate adenylylation/deadenylylation control of glutamine synthetase from photosynthetic organisms have given negative results; we have reexamined the enzyme of Rhodopseudomonas capsulata in this connection. Studies on other systems have shown that adenylylated and deadenylylated forms of glutamine synthetase respond differently to Mg2+ in the γ‐glutamyltransferase assay, and this was observed with the R. capsulata enzyme when its adenylylation state was stabilized by addition of cetyltrimethylammonium bromide to cells before they were harvested for preparation of extracts. The relative activities of glutamine synthetase in the presence and absence of added Mg2+ is an index of adenylylation state and on the basis of this and other criteria, the enzyme in R. capsulata cells grown with excess NH+4 as N source is largely adenylylated, whereas the deadenylylated form predominates when the organism is grown on N2. The results of experiments on the effects of snake venom phosphodiesterase on response of partially purified glutamine synthetase (from R. capsulata cells grown on NH+4) to metal ion supplementation (Mg2+ and Mn2+) provided confirmatory evidence for adenylylation. Conditions are described for demonstration of regulation of R. capsulata glutamine synthetase adenylylation state, in vivo, by light intensity. When the N source for growth is glutamate, the extent of adenylylation decreases with increase of light intensity. In cells subjected to a sudden shift‐down in light intensity, the extent of glutamine synthetase adenylylation increases rapidly; conversely, a shift‐up in light intensity causes, after a lag, a decrease in the degree of adenylylation. The light intensity effects are interpreted to reflect regulation of adenylylation/deadenylylation by the ‘energy state’ of the cell.
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.
Enzymatic Incorporation of ATP and CTP Analogues into the 3′ End of tRNA
Authors: SPRINZL Mathias , STERNBACH Hans , VON DER HAAR Friedrich , CRAMER Friedrich
Published: March 2005

Structural analogues of adenosine 5′‐triphosphate and cytidine 5′‐triphosphate were investigated as substrates for ATP(CTP): tRNA nucleotidyl transferase. Eight out of 26 ATP analogues and six out of nine CTP analogues were incorporated into the 3′ terminus of tRNA. In general, for the recognition of the substrates the modification of the cytidine is less critical than is the modification of adenosine. An isosteric substitution on the ribose residue is possible in both CTP and ATP. The free hydroxyls of these triphosphates can be replaced by an amino group or hydrogen atom without loss of substrate properties. Modifications of positions 1, 2, 6 and 8 on the adenine ring of ATP are not allowed whereas modification on positions 2, 4 and 5 on the cytosine ring of CTP are tolerated by the enzyme. No differences can be observed in the substrate properties of ATP(CTP): tRNA nucleotidyl transferase isolated from different sources. Methods for preparation of tRNA species, which are shortened at their 3′ end by one or more nucleotides, and analytical procedures for characterisation of these modified tRNAs are described.
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.
The 3′‐Terminal Nucleotide Sequence of Encephalomyocarditis Virus RNA
Authors: MERREGAERT Josef , VAN EMMELO John , DEVOS René , PORTER Alan , FELLNER Peter , FIERS Walter
Published: March 2005

Poly(A)‐containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA‐dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA‐sequencing methods. The complement corresponds to the 3′ end of encephalomyocarditis RNA. The hexanucleotide (5′‐3′)(A‐A‐U‐A‐A‐A) found in this sequence is also present in all 3′ non‐coding regions of poly(A)‐containing eukaryotic mRNAs studied until now, in nearly identical positions relative to the poly(A) tail. The possible biological significance of this structural homology is discussed.
Ribosomal Proteins of HeLa Cells
Authors: SCHIFFMANN Dietmar , HORAK Ivan
Published: March 2005

Ribosomal proteins from HeLa cells were analyzed by two‐dimensional polyacrylamide gel electrophoresis (Kaltschmidt–Wittmann) and dodecylsulfate polyacrylamide gel electrophoresis (Laemmli). 35 proteins are associated with the small ribosomal subunit and 47 proteins with the large ribosomal subunit. The HeLa ribosomal proteins S6, S32, L40b,c, L41 and L42 are phosphorylated in vivo and in vitro. Minor differences between HeLa and rat liver ribosomal proteins were revealed by their direct coelectrophoresis.
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.
Determination of ADP‐ribose and Poly(ADP‐ribose) by a New Radioimmunoassay
Authors: BREDEHORST Reinhard , FERRO Ari M. , HILZ Helmuth
Published: March 2005

A specific and sensitive radioimmunoassay for ADP‐ribose has been developed on the basis of the selective conversion of ADP‐ribose to 5′‐AMP by alkaline treatment. Antibodies highly specific against 5′‐AMP allowed quantification of ADP‐ribose converted to 5′‐AMP in the range of 1–40 pmol, and in the presence of large quantities of nucleic acids or 3′‐AMP.

Poly(ADP‐ribose) could also be determined when degraded to ADP‐ribose by poly(ADP‐ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP‐ribose residues after glycohydrolase treatment and of 5′‐AMP from the nonreducing end obtained by phosphodiesterase catalyzed hydrolysis.

The high specificities of the alkaline conversion of ADP‐ribose to 5′‐AMP and of the radioimmunoassay for 5′‐AMP allowed quantification of protein‐bound ADP‐ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples.
Characteristics of a Coupled Cell‐Free Transcription and Translation System Directed by Vaccinia Cores
Authors: PELHAM Hugh R. B. , SYKES Jane M. M. , HUNT Tim
Published: March 2005

A coupled transcription and translation system is described in which protein synthesis is directed by mRNA synthesised in situ by vaccinia virus cores. The cell‐free system is based on a micrococcal‐nuclease‐treated reticulocyte lysate.

The polypeptides made in vitro include many authentic early vaccinia proteins, but also other proteins which were not detected in infected cells.

Concentrations of cores which inhibit host cell protein synthesis in vivo caused a delayed inhibition of translation in vitro; this was partly, but not entirely, due to dsRNA associated with the cores.

The mRNA made was methylated by core enzymes. Inhibition of methylation reduced the rate of translation tenfold; unmethylated RNA bound ribosomes poorly, but was nevertheless translated faithfully.
The Primary Structure of Bovine Pancreatic Phospholipase A2
Authors: FLEER Eduard A. M. , VERHEIJ Hubertus M. , DE HAAS Gerard H.
Published: March 2005

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross‐linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues.

The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids.

Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.
Use of Synthetic Analogs for a Study on the Structure‐Activity Relationship of Apamin
Authors: GRANIER Claude , MULLER Enrique PEDROSO , VAN RIETSCHOTEN Jurphaas
Published: March 2005

[Lys13, Lys14]Apamin, [Lys13]apamin and [Lys14]apamin, three structural analogs of the bee venom neurotoxin, have been obtained by solid‐phase peptide synthesis while an attempt to obtain [Cit13]apamin failed, probably at the step of reoxidation of cysteines. After the chemical purity of these three derivatives had been assessed, further chemical modifications led to three new peptides: [Ac‐Cys1, Lys(Ac)4, Lys(Ac)13]apamin, [Ac‐Cys1, Lys(Ac)4, Lys(Ac)14]apamin and [Har4, Har13, Har14]apamin. These six analogs have been tested for their neurotoxicity, i.e. determination of LD50 for mouse by subcutaneous injection. A lethal potency is observed only when the region 13–14 of the sequence contains a double positive charge. One arginyl residue is necessary for a high biological activity, while its location in position 13 or 14 is of minor importance. When homoarginine (Har) replaces arginyl residues the neurotoxicity is lowered.
Immunochemical Measurement of Conformational Heterogeneity of Poly(Inosinic Acid)
Authors: STOLLAR B. David , DE CLERCQ Erik , DROCOURT Jean‐Louis , THANG Minh Nguy
Published: March 2005

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with antipoly(I) antiserum; (b) their ability to bind to a solid‐phase anti‐poly(I) antibody‐Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double‐stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non‐inducer poly(I)s; the inducers reacted weakly with anti‐poly(I) antiserum and were the only ones that reacted with antibodies to double‐stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non‐inducer poly(I) in quantitative aspects of poly(I) · poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti‐poly(I) antiserum, did not react with anti‐double‐stranded‐RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.
A Universal Lipid Exchange Protein from Rat Hepatoma
Authors: DYATLOVITSKAYA Ella V. , TIMOFEEVA Natalya G. , BERGELSON Lev D.
Published: March 2005

The postmicrosomal protein fraction from rat hepatoma 27 adjusted to pH 5.1 stimulates phospholipid exchange between rat liver microsomes and mitochondria with higher rates and in a less specific way than the corresponding fraction from rat liver. A phospholipid exchange protein has been purified to homogeneity from the hepatoma pH‐5.1 supernatant by gel filtration on Sephadex G‐75 and ion‐exchange chromatography on carboxymethylcellulose. The isolated protein had a molecular weight of 11 200 as determined by electrophoresis on polyacrylamide in the presence of dodecyl sulfate and of 11 168 as calculated from the amino acid composition. Isoelectric focusing showed a single band at pH 5.2.

In the assay system rat liver microsomes → mitochondria the protein exhibits a complete lack of substrate specificity transferring all the major microsomal phospholipids to about the same extent. The possible role of the isolated phospholipid exchange protein in the chemical dedifferentiation of hepatoma cell membranes is discussed.
Brown‐Adipose‐Tissue Mitochondria: Photoaffinity Labelling of the Regulatory Site of Energy Dissipation
Authors: HEATON Gillian M. , WAGENVOORD Rob J. , KEMP Albertus , NICHOLLS David G.
Published: March 2005

Brown‐adipose‐tissue mitochondria possess an energy‐dissipating ion uniport which is inhibited by purine nucleotides. The regulatory nucleotides bind to a high‐affinity site on the outer face of the inner membrane which is independent of the adenine nucleotide translocator. A direct correlation between affinity for the regulatory site and ability to inhibit the ion uniport is demonstrated for a number of nucleotide analogues. 8‐Azido‐adenosine 5′‐triphosphate, a photoaffinity label, also competes with GDP for the binding site and induces respiratory control. 8‐Azido‐adenosine [γ‐32P]triphosphate was prepared and covalently bound to hamster brown‐adipose‐tissue mitochondria by near‐ultraviolet irradiation. Two major radioactive bands were identified of apparent molecular weight 30 000 and 32 000, representing 6% and 10% of the inner membrane protein respectively. Selective labelling enabled the 30 000‐Mr protein to be identified as the carboxyatractylate binding component of the adenine‐nucleotide translocator and the 32 000‐Mr protein to be identified as the regulatory site of the energy‐dissipating ion uniport. The levels of the 32 000‐Mr protein in the inner membrane of guinea‐pig brown‐adipose‐tissue mitochondria correlate with the degree of thermogenic adaptation of the animal.