bluuebunny/crossref_metadata_2025_split huggingface dataset:
Steric Course of the NIH Shift in the Enzymic Formation of Homogentisic Acid
Authors: LEINBERGER Robert , RÉTEY Anos , HULL William E. , SIMON Helmut 
Published: July 2010

4‐Hydroxyphenylpyruvate dioxygenase has been purified from beef liver to a specific activity of 0.021 U/mg at 25°C.
Samples of (3R)‐ and (3S)‐4′‐hydroxyphenyl[3‐2H1]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto—enol‐isomerase (tautomerase). These were converted in situ by the dioxygenase into the corresponding deuterium‐labelled homogentisic acids, which were isolated as the crystalline 3′,5′‐dimethyl derivatives after treatment with dimethyl sulphate.
2′,5′‐Dimethoxy‐(E)‐cinnamate was deuterated by enoate reductase from Clostridium sp. La 1 to afford (2S, 3R)‐3‐(2′,5′‐dimethoxyphenyl)‐[2‐2H1, 3‐2H1]propionate which was in turn chemically degraded to (S)‐2,5‐dimethoxyphenyl[2H1]acetic acid.
After the chiroptical characterisation of the chiral 2,5‐dimethoxyphenyl[2H1]acetic acid had failed, the following samples of 2,5‐dimethoxyphenylacetic acids were converted into the (–)‐menthyl ester: (a) unlabelled, (b) statistically deuterated in position 2 (and in the aromatic positions), (c) (2S)‐[2‐2H1] as reference, (d) produced by the dioxygenase from (3R)‐4′‐hydroxyphenyl[3‐2H1]pyruvate, and (e) produced by the dioxygenase from (3S)‐4′‐hydroxyphenyl[3‐2H1]pyruvate.
In the 500‐MHz 1H‐NMR spectrum the 2‐methylene protons of unlabelled 2,5‐dimethoxyphenylacetic acid (–)‐menthyl ester appeared as a well resolved AB system. Using a triple resonance technique and the reference samples (b) and (c) the 2‐HRe atom could be correlated with the high‐field half and the 2‐HSi atom with the lowfield half of the AB system.
By comparison of the spectra of the enzymically obtained samples (d) and (e) with the reference spectra it was concluded that in the dioxygenase reaction the side‐chain migration occurred with retention of configuration at the methylene C atom.
Compositional Heterogeneity of the Chloroplast DNAs from Euglena gracilis and Spinacia oleracea
Authors: SCHMITT Jürgen M. , BOHNERT Hans‐Jürgen , GORDON Karl H. J. , HERRMANN Reinhold , BERNARDI Giorgio , CROUSE Edwin J. 
Published: July 2010

The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using several different experimental approaches.Euglena chloroplast DNA has a dG + dC content of28 %. Preparations averaging 20 × 106 in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT‐rich sequences must occur in short stretches interspersed with dG + dC‐rich stretches.Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, buoyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30 of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.
Crystallization and Properties of Carboxypeptidase Aγ from Porcine Pancreas
Authors: KOIDE Atsushi , YOSHIZAWA Masayuki , KURACHI Kotoku 
Published: July 2010

Carboxypeptidase Aγ from porcine pancreas was purified to homogeneity by ammonium sulfate fractionation, autolysis, batch absorption and elution from DEAE‐Sephadex, and crystallization. The overall purification was about 32‐fold with a yield of 31% and the specific activity of the purified protein was 108 units/mg protein.The apparent relative molecular mass determined by gel filtration on a Sephadex G‐200 column was 38900. The amino‐terminal sequence of the porcine carboxypeptidase Aγ was Asn‐Tyr‐Ala‐Thr‐Tyr‐His‐Thr‐Leu‐GluGlu‐Ile‐Tyr‐Asp‐Phe‐Met‐Asp‐Ile‐Leu‐Val‐Ala‐Glu‐His‐Pro‐Gln‐Leu‐ which was highly homologous to that of bovine carboxypeptidase AYγ. The purified enzyme was characterized with respect to isoelectric point (4.3). Km for Nα‐carbobenzoxyglycyl‐l‐phenylalanine (Cbz‐Gly‐lPhe) (20 mM), amino acid composition, pH optimum, pH stability, stability at different temperatures and effect of drying. The enzyme contained 1.01 mol zinc/mol and was inhibited by chelating agents such as iEDTA and o‐phenanthroline.Among substrates such as Cbz‐Gly‐lPhe, Nα‐benzoylglycyl‐l‐arginine, various kinds of amino acid esters, casein and elastin, porcine carboxypeptidase Aγ showed an enzymatic activity only towards Cbz‐Gly‐lPhe and casein. These data are in good agreement with the substrate specificity of bovine carboxypeptidase A.
Transcription of the Cloned Genes for Ribosomal 5‐S RNA in a System Reconstituted in vitro from HeLa Cells
Authors: GRUISSEM Wilhelm , KOTZERKE Michael , SEIFART Klaus H. 
Published: July 2010

Cytoplasmic extracts from HeLa cells, containing transcription factors and intact RNA polymerase C, were used to transcribe the cloned genes for ribosomal 5‐S RNA from Xenopus borealis. It was found that the endogenous enzyme in these extracts synthesizes discrete products of 5‐S size which hybridize predominantly to the 5‐S RNA gene region and reveal an oligonucleotide pattern characteristic of authentic 5‐S ribosomal RNA. The overall transcription of the plasmic DNA is greatly repressed in the presence of the cytoplasmic extract. The specific production of 5‐S RNA by the cytoplasmic extract depends on an optimal DNA concentration, beyond which the excess DNA is transcribed unspecifically. The crude extracts contain a factor which can be enriched by chromatography on phosphocellulose and which binds to DNA. In the presence of the cytoplasmic extract, this factor significantly stimulates 5‐S RNA synthesis at high DNA concentration. No stimulation is observed at limiting DNA concentrations, already saturated by endogenously contained factor. The synthesis of 5‐S RNA in this system depends on the appropriate template since it is not observed with the cloned genes for 5‐S from Drosophila.The cytoplasmic extracts endogenously contain a limited capacity to transcribe the genes for ribosomal 5‐S RNA. This process can be stimulated significantly by the addition of optimal concentrations of purified RNA polymerase C, which by itself transcribes these genes in an entirely random fashion. Reconstitution of plasmid DNA with the DNA‐binding protein and purified polymerase does not lead to the production of 5‐S RNA. From these data we conclude that the cytoplasmic extracts contain additional factor(s) which are deficient in the purified polymerase molecule and which are required together with the DNA‐binding protein for the specific synthesis of ribosomal 5‐S RNA.The observed stimulation of 5‐S RNA synthesis is not seen with RNA poiymerase C from yeast cells, indicating an evolutionary divergence of functional significance in the polymerase molecule.
Citrate‐Dependent Iron Transport System inEscherichia coliK‐12
Authors: HUSSEIN Saber , HANTKE Klaus , BRAUN Volkmar 
Published: July 2010

Induction of the citrate‐dependent iron transport system ofEscherichia coliK‐12 required 0.1 mM citrate and 0.1 μM iron in the growth medium. Five‐ten‐times more iron than citrate was taken up into the cells which suggests that citrate was largely excluded from the transport. Fluorocitrate and phosphocitrate induced the citrate, dependent iron transport system although they supported iron uptake only very poorly. An outer membrane protein (fecA), belonging to the transport system, was induced infecBmutants which were devoid of citrate‐dependent iron transport. The intracellular citrate and iron concentrations were 10‐100‐times higher than the external concentrations required for induction of the transport system. It is concluded that only exogenous ferric citrate induced the transport system, and that citrate did not have to enter the cytoplasm.The Γn10 transposon, conferring tetracycline resistance, was inserted near the fec gene region which controls the expression of the citrate‐dependent iron transport system. The determination of the cotransduction frequencies of Tn10 with thefecAandfecBmarkers suggested the gene orderfecA,fecBΓn10.
Fluorimetric Study of Yeast tRNAPheCCF in the Complex with Phenylalanyl‐tRNA Synthetase
Authors: LEFEVRE Jean‐François , BACHA Hassen , RENAUD Michel , GANGLOFF Jean , REMY Pierre , EHRLICH Ricardo , VON DER HAAR Friedrich 
Published: July 2010

The fluorescence properties of yeast tRNAPheCCF (tRNAPhe in which the 3′‐terminal adenosine has been replaced by formycin) and tRNAPheCCFoxi‐redafter periodate oxidation followed by borohydride reduction) were studied in the complex with the cognate ammoacyl‐tRNA synthetase. In both cases a conformational change affecting the 3′ end was observed in a magnesium concentration range close to 1 mM. The modification of formycin fluorescence could be ascribed simultaneously to the existence of a tautomeric equilibrium of the fluorescent probe and to a pH effect arising from a prototropic effect at the active site of phenylalanyl‐tRNA synthetase, and to a partial destacking of the 3′‐formycin from the adjacent C residue.The observed transconformation, which can be related to the structure modification of the anticodon loop previously reported [Ehrlich, Lefevre, and Remy (1980) Eur. J. Biochem. 103, 145–153], takes place in the magnesium concentration range allowing the transfer of the activated amino acid from the adenylate to the tRNA.The interconnection between the anticodon loop and the accepting end was further supported by the observation that wybutine excision hinders the specific structure modification of 3′‐formycin upon binding to the synthetase.The tRNAPhe transconformations occurring in the complex with the cognate synthetase probably reflect a reciprocal adaptation of both macromolecules which might lead to the optimal aminoacylation velocity and thus contribute to the specificity of aminoacylation. since it was previously established that this specificity relies more strongly on the kinetics of the reaction than on a discrimination of tRNAs according to different affinities [Ebel et al. (1973) Biochimie Paris 55, 547–557].
Fast Stages of Photoelectric Processes in Biological Membranes
Authors: DRACHEV Lei A. , SKULACHEV Vladimir P. , KAULEN Andrey D. , KALAMKAROV Grigory R. , OSTROVSKY Michad A. 
Published: July 2010

The functioning of visual rhodopsin as a photoelectric generator has been demonstrated with a direct method. Photoreceptor discs were incorporated into a phospholipid‐impregnated collodion film. Illumination of the resulting system with continuous light was found to induce formation of an electric potential (the disc‐free side positive) that was measured with two electrodes separated by the film. A photopotential exceeding 40 mV was shown. It dissipated before the light source was switched off. A 15 ns 530‐nm laser flash induced the formation of a photopotential of up to 35 mV whose appearance was preceded with a small oppositely directed electrogenic phase. This ‘negative’ photoresponse took less than 200 ns. The ‘positive’ photoresponse was composed of at least two phases (t1/2about 500 μs and several milliseconds). The latter was shown to correlated with formation of metarhodopsin II.A 347‐nm laser flash added after a 530‐nm flash resulted in a photoelectric effect similar to that initiated by 530‐nm flash but of opposite direction. The 347‐nm response was completely abolished by hydroxylamine preventing the accumulation of metarhodopsin II. The response at 530 nm proved to be hydroxylamine‐resistant. Both the amplitude and the decay time of the flash‐induced potential were maximal in the response to the first flash, each subsequent flash being less effective than the preceding one. Flashes were found to cause acceleration of the photopotential decay. The latter effect proved to be due to a increase of membrane conductance that developed faster than in 50 ms. Addition of 11‐cisretinal after illumination improved the amplitude of the photoresponse but not the conductance. The light‐induced increase in conductance was insensitive to hydroxylamine.It is suggested that a function of visual rhodopsin consists in generating a potential difference across the photoreceptor disc membrane which responds with a increase in membrane permeability to a rise of the membrane potential. A possible role of an electric breack‐down of the membrane, induced by the rhodopsin‐generated local or partially delocalized electric field has been discussed.
Characterization of a Structure Close to the Coenzyme‐Binding Site of Liver Aldehyde Dehydrogenase
Authors: von BAHR‐LINDSTRÖM Hedvig , JÖRNVALL Hans , SOHN Sigrid , WOENCKHAUS Christoph , JECK Reinhard 
Published: July 2010

NAD analogues with the nicotinamide moiety exchanged for acetylpyridinio‐pentyl or acetylpyridinio‐butyl groups function as coenzymes in the enzymatic reaction with liver aldehyde dehydrogenase. The corresponding bromoacetyl derivatives bind to the coenzyme‐binding site of the enzyme and inactive the protein by covalent modification of single residues close to the active site.Protection by coenzymes and substrate against the inactivation differs slightly for the two coenzyme analogues, suggesting the presence of more than one reactive residue. This is consistent with the results of differential carboxymethylations of cysteine residues of the basic isozyme in the presence and absence of the inhibitor disulfiram.The amino acid sequence around one reactive cysteine residue close to the active site of the acidic isozyme was determined after labeling with the butyl coenzyme analogue. This structure bears no extensive homology to corresponding known structures of dehydrogenases working on other types of aldehyde substrates.
Protein Synthesis in Brine Shrimp Embryos
Authors: WOODLEY Charles L. , ROYCHOWDHURY Mahendra , OLSEN Kenneth W. , WAHBA Albert J. , MACRAE Thomas H. 
Published: July 2010

We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co‐eIF‐2(A) and Co‐eIF‐2, (B). These factors are essential for ternary complex formation and binding of [35S]‐Met‐tRNAf to 40‐S ribosomal subunits with 15–30 μg eIF‐2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co‐eIF‐2(A) is composed of a single polypeptide of Mr 65000, whereas Co‐eIF‐2(B) contains polypeptides of Mr 105000 and 112000.Co‐eIF‐2(A) is sensitive to 4.5 μM aurintricarboxylic acid but Co‐eIF‐2(B) requires approximately 15 μM aurintricarboxylic acid to give 50% inhibiton of ternary complex formation. The stimulatory activity of both actors is abolished by pretreatment of the proteins with N‐ethylmaleimide. Artemia eIF‐2 rapidly binds [3H]GDP or [3H]GTP and at 15°C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) have no effect on the exchange or the amount of nucleotide bound.The small subunit (Mr 43000) of Artemia eIF‐2 is phosphorylated in the presence of the rabbit reticulocyte heme‐repressible kinase. Tryptic digestion of [32P]phosphorylated eIF‐2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF‐2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF‐2 is active in ternary complex formation, in AUG‐dependent binding of [35S]Met‐tRNAf to 40‐S ribosomal subunits and in cell‐free protein synthesis. Both Co‐eIF‐2(A) and Co‐eIF‐2(B) stimulate ternary complex formation with phosphorylated eIF‐2. A kinase that phosphorylates the small subunit of eIF‐2 is present in the postribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos.
Relationship between Membrane Permeability and the Translation Capacity of Human HeLa Cells Studied by Means of the Ionophore Nigericin
Authors: ALONSO Miguel A. , CARRASCO Luis 
Published: July 2010

Treatment of HeLa cells with the ionophore nigericin causes a drastic inhibition of protein synthesis which is dependent on the concentration of monovalent ions present in the medium. 0.7 μM nigericin inhibits protein synthesis by almost 100% in a culture medium containing 5 mM KCl and 40 mM NaCl while no inhibition is observed with similar concentrations of nigericin in a medium containing 50 mM KCl and 25 mM NaCl. Moreover, once the inhibition of protein synthesis by the action of the ionophore has occurred in the former medium, it can be readily reversed to control levels by placing the cells in a medium with the appropriate concentration of monovalent ions, even though nigencin is still present. These results do not support the idea that the inhibition of translation by nigericin in intact cells involves the stable modification of the protein‐synthesizing machinery, nor even that translation inhibitors are generated by the action of nigericin on the cell membrane.The hypothesis that the blockade of translation by nigericin is produced by the modification of the monovalent ion concentration in the cell cytoplasm is reinforced in this work by several lines of evidence. Firstly, a striking parallelism between the inhibition of protein synthesis and the modification of the 86Rb+ content of HeLa cells was detected. Secondly, no variation in the cellular ATP content was observed at concentrations of nigericin that drastically blocked protein synthesis, and thirdly, no inhibition of the uptake of the radioactive amino acid precursor took place. Several ionophores, mainly A 23187, monensin, gramicidin D and valinomycin, all reduced both the translation capacity and the levels of 86Rb+ ions in cultured HeLa cells.These results reinforce the idea that macromolecular synthesis in mammalian cells might be regulated by membrane‐mediated events that in turn influence the concentration of monovalent ions in the cell.
Interaction of the N‐Terminal and C‐Terminal Domains of Elongation Factor G on Formation of Complexes with Guanyl Nucleotides
Authors: KASHPAROV Ivan A. , SEMISOTNOV Gennady V. , ALAKHOV Yuly B. 
Published: July 2010

Polarized fluorescence studies of interaction between guanyl nucleotides (GTP and GDP) and elongation factor G and its N‐terminal tryptic fragment T*2, carrying a fluorescent group (aminorhodamine B) at the exposed cysteine residue, has shown that binding of nucleotides by an intact EF‐G molecule at neutral pH essentially affects the mobility of the fluorescent group. GTP binding changes its relaxation properties to a greater extent than GDP binding. At the same time it was demonstrated that the spectrum of relaxation times of the fluorescent group practically does not change on binding of nucleotides by the N‐terminal fragment T*2 (in the absence of the C‐terminal uomain) or in the case when the three‐dimensional structure of the intact EF‐G molecule is destabilized (pH 10). Comparison of the relaxation properties of EF‐G and its N‐terminal fragment T*2, carrying a fluorescent group at the exposed cysteine residue, at pH 7.5 and 10, indicates that the C‐terminal domain is involved in the formation of the close environment of the exposed cysteine residue located in the N‐terminal part of EF‐G.A conclusion is drawn on the nucleotide‐induced influence of the C‐terminal domain on a change of the exposed cysteine residue environment on guanyl nucleotide binding with EF‐G at neutral pH and a hypothetical model of the EF‐G molecule is proposed.
5,6‐Dichlororibofuranosylbenzimidazole Inhibits the Rate of Transcription Initiation in Intact Chironomus Cells
Authors: EGYHÁZI Endre , PIGON Andrew , RYDLANDER Lars 
Published: July 2010

The effect of nucleoside analogue 5.6‐dichloro‐l‐β‐D‐ribofuranosylbenzimidazole on RNA transcription has been reinvestigated after the observation that explanted salivary gland cells of Chironomus tentans efficiently take up of exogeneous ATP in an undegraded form. We addressed the question of whether the inhibitory action is exerted predominantly by accentuation of premature termination of hnRNA transcripts or by inhibition of the initiation step. Electrophoretic analyses in 10% polyacrylamide gels of [α‐32P]ATP‐labelled low‐molecular‐weight RNA chains in the 30–1000‐nucleotide range did not reveal the accumulation of early terminated transcripts irrespective of whether dichlororibosylbenzimidazole was present in the medium or not. The separation of 70% ethanol‐soluble α‐32P‐labelled material by thin‐layer electrophoresis led to a similar result: no detectable accumulation of very short transcripts (oligonucleotides) due to a possible premature termination of transcription. A selective labelling of newly initiated RNA transcripts was achieved by incorporation of 32P from [β‐32P]ATP into the 5′ termini of RNA molecules which start with ATP.The internal uptake of 32P was low, if any, as measured by the sensitivity of RNA labelling aganist alkaline phosphatase. In addition to a significant portion of β‐32P‐labelled hnRNA chains, labelling of newly initiated preribosomal 38‐S RNA, and 75‐S RNA of Balbiani ring origin, is demonstrated as well. Incorporation of label into 38‐S and 75‐S RNA exhibits the same kind of specificity against dichlororibosylbenzimidazole as when isotopic nucleosides are used for RNA labelling: i.e. the formation of labelled 38‐S RNA is resistant while that of 75‐S RNA is sensitive. During a 5‐min pulse, in the presence of the analogue, the rate of accumulation of newly initiated β‐32P‐labelled transcripts, predominantly hnRNA, was inhibited by more than 60%. The data taken together strongly indicate that the inhibition of the transcription of hnRNA genes with 5,6‐dichloro‐ ribofuranosylbenzimidazole in Chironomus takes place at the level of initiation and argues against the existence of prematurely terminated hnRNA transcripts
Preparation and Characterisation of Homogeneous Neurotoxin Type A fromClostridium botulinum
Authors: TSE Chun K. , DOLLY J. Oliver , HAMBLETON Peter , WRAY Dennis , MELLING Jack 
Published: July 2010

Large‐scale production and purification of complexes betweenClostridium botulinumneurotoxin and haemagglutinin have been achieved.Haemagglutinin‐free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography. onp‐aminophenyl β‐D‐thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE‐Sephacel.The resultant neurotoxin was homogeneous on isoelectric focussing (PI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non‐reducing conditions when its Mrwits 1.4 × 105; after reduction two polypeptides (Mr= 9.9 and 5.5 × 104) were present.On double‐ininiunodiffusion gels, using antiserum against neurotoxin‐haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non‐toxic protein (Mr= 1.3 × 105). which co‐purifies with the neurotoxin but is removed by the ion‐exchange chromatography step.Application of the neurotoxin to animtilsin vitroorin vivoproduced near complete and irrevenible blockade of neurotransniission. Botulitiisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end‐plate potentials was altered from the normal ‘bell‐shaped’ to II skewed distribution.In normal muscle, a large transient increase in frequency of the miniatures was produced by β‐bungaro‐toxin. In contrast. with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition. the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of. muscle modifies the action of β‐bungarotoxin.