bluuebunny/crossref_metadata_2025_split huggingface dataset:
Significance of Ribonucleotide Reduction in the Biosynthesis of Deoxyribonucleotides in Escherichia coli
Authors: Karlström O. , Larsson A. 
Published: March 2005

A pyrimidine auxotroph of Escherichia coli, possessing decreased deaminase activities for cytidine and deoxycytidine, was grown with uniformly 14C‐labeled cytidine or uridine as sole pyrimidine source. The incorporation of radioactivity into the individual mononucleotides of DNA and RNA was determined. The pyrimidine nucleotides were degraded, and the distribution of isotope between their pentose and base moieties was analyzed.[14C]‐Cytidine was incorporated into RNA without loss of its isotope content. Furthermore, CMP of RNA and deoxyCMP of DNA had identical specific activities and pentose: base ratios. Therefore deoxyCMP of DNA was formed exclusively by reduction of a cytidine derivative.Only approximately 20% of the pentose isotope content of [14C]‐uridine was retained after incorporation into RNA. CMP, UMP, deoxyCMP, and deoxyTMP isolated from the nucleic acids had approximately equal specific activities and pentose: base ratios, indicating that both pyrimidine deoxyribonucleotides were formed by ribonucleotide reduction.[14C]‐Cytidine was converted to UMP of RNA with almost complete loss of isotope from the ribosyl moiety, but was converted to deoxyTMP of DNA with little loss of label. After estimation of different enzymatic activities in cell‐free extracts it was concluded that the major pathway yielding deoxyTMP of DNA did not involve deoxyTMP synthetase but rather proceeded directly from a deoxycytidine compound.
Initiation of DNA‐Dependent RNA Synthesis and the Effect of Heparin on RNA Polymerase
Authors: Walter G. , Zillig W. , Palm P. , Fuchs E. 
Published: March 2005

The kinetics of RNA synthesis, catalyzed by DNA‐dependent RNA polymerase from Escherichia coli, show a lag phase at high ionic strength or at low temperature when using a double‐stranded DNA as template. This lag phase increases with decreasing temperature at constant ionic strength or with increasing ionic strength at constant temperature. With single‐stranded DNA as template, no lag phase is observed. The length of the lag phase is greatly shortened by pre‐incubation of the enzyme with DNA at low Mg++ concentration, independent of the presence of substrate.Heparin is a strong inhibitor of free and DNA‐bound polymerase. Two heparin molecules are required for the inactivation of one enzyme molecule. Enzyme engaged in RNA synthesis is instantly inhibited on single‐stranded DNA but not on double‐stranded DNA. With the aid of this inhibitor it is shown that 10 minutes after the start of incubation, at 16°, all enzyme molecules have initiated RNA chains on double‐stranded DNA.
Thermodynamique des associations de poly A et poly U en milieu neutre et alcalin
Authors: Massoulié J. 
Published: March 2005

The complexes of poly A and poly U are interesting models for studying the structure of nucleic acids. In the first part of this paper, the thermal stability of these complexes has been studied as a function of both pH and ionic strength in alkaline solution. The thermal stability is measured by the half transition temperatures, or Tm's. There are four different kinds of transitions: Tm2–1 refers to the dissociation of the two‐stranded complex, poly (A + U); Tm2–1 to the dissociation of the three‐stranded complex, poly (A + 2U) [4]. Tm2–2 is the temperature of half dissociation of poly (A + 2U) into poly (A + U) [1], and Tm2–2 is the temperature of rearrangement of 2 poly (A + U) into poly (A + 2U) [3] (Fig. 1). It is found that Tm2–1, and Tm3–1 decrease as pH increases above neutrality (Fig.2 and 3), but that Tm2–2 does not vary (Fig.3). This effect is readily explained if it is assumed that the ionisation of uracil brings a change in the state of the free poly U, but that the complexes themselves are not modified.In the second part of the paper a simple thermodynamic formulation is given which represents satisfactorily the relationship between the four equilibria, as observed at neutral pH (Fig.1). A more complex treatment is then presented. It includes quantitation of electrostatic interactions, according to the theory of Kotin [18] and of the free enthalpy term corresponding to the organisation (stacking) of poly A. This leads to an excellent representation of the experimental data (Fig.5).In alkaline solution, one can calculate the free enthalpy change associated with the ionisation of poly U at the Tm. According to our observations, it is likely that this is the only factor responsible for the decrease of Tm. Therefore, we can write that the sum of the free enthalpy of complex formation in the same conditions, but at pH 7, and of the free enthalpy of ionisation is zero. This leads to a determination of ΔG both as a function of temperature and ionic strength: the variation of ΔG with these factors is shown to be in good agreement with our detailed model. However, the absolute values are different, probably because of uncertainties concerning the data for poly A stacking enthalpy and entropy.Apart from providing evaluation of the thermodynamic parameters for double and triple helix formation, our results lead us to emphasize the importance of the thermodynamic state of the unassociated polynucleotides. This may be particularly relevant when one considers the partially complementary secondary structures of RNA's.
On the Structure and Function of Reduced Nicotinamide Adenine Dinucleotide Phosphate‐Cytochrome f Reductase of Spinach Chloroplasts
Authors: Forti G. , Sturani E. 
Published: March 2005

NADPH‐eytochrome f reductase from spinach chloroplasts has been purified, and its amino acid composition is reported.
The specificity of the enzyme for different electron acceptors has been studied. In addition to higher plant cytochrome f, Euglena gracilis cytochrome f and plastocyanin are reduced. The reaction with these acceptors occurs through a two‐step mechanism, whereas higher plant cytochrome f forms a ternary complex with NADPH and the enzyme. The Km values for NADPH in the reactions with the different electron acceptors, the Km's for the electron acceptors and the turnover numbers are reported.
The sulphydryl reagents slowly inactivate the enzyme and enhance the fluorescence of enzyme‐bound FAD and of the tryptophan residues of the protein. In the presence of NADPH, the inactivation is very rapid, and FADH2 is split from the apoenzyme. Preincubation of the enzyme with NADPH inactivates reversibly all the reactions except cytochrome f reductase.The results are interpreted in terms of structure to function relationships and the significance of the NADPH‐cytochrome f reductase in photosynthetic electron transport is also discussed.
Préparation et propriétés de dérivés poly‐DL‐alanylés d'un inhibiteur de la trypsine (inhibiteur de Kunitz et Northrop)
Authors: Acher R. , Chauvet J. , Arnon R. , Sela M. 
Published: March 2005

The basic trypsin inhibitor of bovine pancreas (Kunitz and Northrop inhibitor) has been alaninated with N‐carboxy‐DL‐alanine anhydride in a 0.05 M phosphate buffer, pH 7, containing 20% (v/v) dioxane.When the molar ratio anhydride/inhibitor is 10 and the reaction is performed during 1 hour, approximately 70% of the α‐amino group are blocked but the four ∈‐amino groups are only weakly affected. The derivative has an inhibiting capacity similar to that of the native inhibitor. The performic acid oxidized product is subjected to tryptic hydrolysis and the peptides are separted by the fingerprinting technique, then analyzed. The detected peptides are simolar to those given by the oxidized inhibitor execept for the N‐terminal fragment which has about 2 additional alanine residues presumably on the α‐amino group.When the molar ratio anhydride/inhibititor is 250 and the reaction is performed during 74 hours, the five amino groups are fully blocked. The derivative is virtually inactive. The oxidized product, split by trypsin, gives a peptide map from which the lysyl peptides are absent. In contrast junction peptides can be detected and analyzed. The average number of alanine residues fixed on each lysine has been determined. The four lysines react with N‐carboxy‐DL‐alanine anhydride in a similar manner. When the four lysines of the inhibitor are converted to homoarginine residues by guanidination, the activity remains, but when polyalanine chains (5–10 residues) are attached to these lysines, the activity is virtually lost. The results suggest that one or several ∈‐amino groups are involved in the interaction between inhibitor and trypsin.
Incorporation spécifique de la L‐[U‐14C] isoleucine dans le groupement hydroxy‐2 méthyl‐2 butyroyle de la glaucarubinone
Authors: Moron J. , Polonsky J. 
Published: March 2005

2‐Hydroxy‐2‐methylbutyric acid (III) occurs esterified to glaucarubolone (II) in glaucarubinone (I), the bitter principle of Simaruba glauca (Simarubaceae).No experimental results concerning the biosynthesis of this acid have yet been reported.The biogenetic precursor of 2‐hydroxy‐2‐methylbutyric acid might be either mevalonic acid, acetoacetate, or isoleucine.We have shown previously that mevalonic acid is not incorporated into the 2‐hydroxy‐2‐methylbutyrate component of glaucarubinone (I) although it is into glaucarubolone (II).We now report our feeding experiments with two batches of young shoots of Simaruba glauca using l‐[U‐14C]isoleucine and sodium [1‐14C]acetate.After 8 days the bitter principles were isolated and the total incorporation of radioactivity determined. The radioactive glaucarubinone (I) was hydrolysed to glaucarubolone (II) and 2‐hydroxy 2‐methyl butyric acid (III). This acid, when obtained from glaucarubolone after incorporation of acetate, has been found to be weakly labelled. In contrast, it carries essentially all the activity of glaucarubinone, when isolated after incorporation of isoleucine.These results establish the specific incorporation of isoleucine into the 2‐hydroxy 2‐methyl butyroyl part of glaucarubinone.
The Regulation of Isoleucine‐Valine Biosynthesis in Saccharomyces cerevisiae
Authors: de Robichon‐Szulmajster H. , Magee P. T. 
Published: March 2005

A study of threonine deaminase from Saccharomyces cerevisiae has been undertaken. The enzyme has been purified 50‐fold, with a low resolution of pyridoxal phosphate coenzyme. The purified enzyme is very unstable under all conditions tried so far. Properties have been studied with crude extracts. Optimum pH has been found to be 8.2. Isoleucine has complex effects on the enzyme activity. At a concentration of 0.1 mM, it is able to stimulate the enzyme. At higher concentrations, isoleucine inhibits. The Ki values increase with increasing pH, but 100% inhibition can be reached at all ranges of pH studied. Affinity of the enzyme for its substrate is even more strongly modified by pH.a) Substrate cooperativity, nil at pH 7, intermediate at pH 8, seems to be maximum at pH 9. At any pH, the substrate cooperativity can be suppressed by addition of L‐valine. In addition, rather low concentrations of isoleucine also act partly as valine does.b) Apparent Km values, measured in the presence of valine, vary with pH in the opposite direction to Ki values. Hill's coefficients for substrate and inhibitor reflect these findings. For threonine alone, they vary from 1.2 at pH 7 to 3 at pH 9. In the presence of valine, they remain close to 1, at all pH. Isoleucine, 0.1 mM, produces an incomplete effect compared to valine. For isoleucine inhibition, the cooperativity coefficients remain close to 3 at all pH. The molecular weight of the enzyme has been determined to be roughly 1.2 × 105, at pH 6.8 and at pH 8.7. Thermal inactivation is unimodal and fails to show desensitization of the enzyme in any of the conditions tried.The implications of these results on the structure of the enzyme have been discussed.
Control of Succinate Oxidation by Succinate‐Uptake by Rat‐Liver Mitochondria
Authors: QQUAGLIARIELLO E. , PALMIERI F. 
Published: March 2005

The uptake of succinate by mitochondria and the rate of respiration in the presence of succinate plus rotenone have been analysed.

In conditions in which Vmax is the same, the apparent Km for succinate is lower when respiration is activated by ion uptake than it is in the active state. The highest value is obtained in the presence of 2,4‐dinitrophenol.
Succinate uptake is maximum during ion uptake, is lower in the active state and lowest in the uncoupled state.
With increasing concentrations of uncoupler, a decrease in the intramitochondrial concentration of succinate is observed parallel to the decrease in respiration.
The inhibition of respiration caused by gramicidin under certain conditions is accompanied by a decrease in the intramitochondrial concentration of succinate.
Citrate and other substrate anions are shown to be competitive with succinate with respect to both the oxidation and the uptake of succinate.
The inhibition of respiration by citrate is less pronounced in particles than in mitochondria.
It is concluded that the rate of respiration with succinate plus rotenone as substrate is controlled by succinate uptake.
Die Biosynthese von 3‐Hydroxymethylpyridin bei Mycobacterium bovis Stamm BCG
Authors: GROSS D. , FEIGE A. , SCHÜTTE H. R. , ZURECK A. 
Published: March 2005

Mycobacteria are able to produce nicotinic acid and 3‐hydroxymethylpyridine (pyridine‐3‐carbinole). Experiments using growing cells of Mycobacterium bovis strain BCG showed that applied 14C‐labelled nicotinic acid is utilized to form radioactive 3‐hydroxymethylpyridine. The resulting incorporation rate was comparatively high. On feeding resting cells with 3‐hydroxymethyl‐[4,6‐14C]pyridine, the reverse reaction was also noticed.Isolated end‐products were degraded to estimate the isotope distribution. The radioactivity of applied [carboxy‐14C]nicotinic acid was almost entirely found in the carbinolic moiety of the isolated pyridine‐3‐carbinole, whereas the label of administered 3‐hydroxymethyl‐[4,6‐14C]‐pyridine was localized in the pyridine nucleus of the isolated nicotinic acid.Results obtained from these feeding experiments showed a direct biogenetic relationship between nicotinic acid and pyridine‐3‐carbinole.
Biochemical Studies on Lipopolysaccharides of Salmonella R Mutants
Authors: DRÖGE W. , RUSCHMANN E. , LÜDERITZ O. , WESTPHAL O. 
Published: March 2005

Analyses performed on the lipopolysaccharides of several Salmonella R‐mutants as well as on their acid degradation products revealed differences in their phosphate content. Two groups of lipopolysaccharides (P+ and P−) could be distinguished, differing by approximately one phosphate group per two heptose units.The additional phosphate group in P+ lipopolysaccharides is linked to the heptosyl‐heptose unit, since heptose phosphate esters could be isolated by the procedure of Slein and Schnell. When degraded P+ polysaccharides were subjected to methylation and methanolysis no peaks of methylated or partially methylated heptose were detected by gas chromatography.Gel filtration on Sephadex revealed that P+ degraded polysaccharides contain heterogenous fractions of higher molecular weight, while the corresponding hydrolysates from P− mutants contain only oligosaccharides of lower molecular weight. Accordingly, P−“lipopolysaccharides” are termed “glycolipids”. It is concluded, that the additional phosphate groups function as diester bridges, thus causing the formation of larger molecules.P+ lipopolysaccharides and P− glycolipids of corresponding chemotypes were found to be distinct serologically when tested by hemagglutination inhibition.
Rapidly Labelled High Molecular RNA from Rat Liver
Authors: SCHÜTZ G. , GALLWITZ D. , SEKERIS C. E. 
Published: March 2005

The rapidly labelled RNA extracted from rat liver homogenate after a 4 min pulse sediments heterogeneously from 10 S to 80 S. After 30 min labelling time an accumulation of radioactivity between 30 S and 50 S is observed. A comparison of these sedimentation patterns of RNA derived from different labelling times indicates a greater metabolic instability of the rapidly labelled heterogeneously sedimenting RNA in relation to that of the 45 S r‐precursor.The RNA sedimenting heavier then 45 S has a base composition which resembles that of DNA. The rapidly labelled RNA is resistant to DNase, pronase and EDTA treatment and thus aggregate formation with protein, DNA and bivalent cations is unlikely.Experiments with actinomycin D and cold orotic acid show a shift of radioactivity to lower sedimentation values which also reflects the metabolic instability of the heavy RNA. Labelling of the ribosomal peaks after actinomycin D treatment could be achieved only after the 30 min pulse, and not after the 4 min pulse, suggesting the absence of an appreciable amount of r‐precursor in the shorter pulse.The RNA extracted from nuclei prepared with Triton X‐100 remained in the upper fractions when analyzed on a sucrose gradient, reflecting the high susceptibility to degrading enzymes.The major part of the rapidly labelled RNA remains associated with the nuclear fraction.
Molecular Weight and Quaternary Structure of Lactic Dehydrogenase
Authors: JAENICKE R. , KNOF S. 
Published: March 2005

Isoenzymes of lactic dehydrogenase from different sources have been reported to show identical molecular weights between 115,000 and 154,000. As compared to the protomer weight of 36,000 this corresponds to association numbers between 3 and 4 suggesting either dissociation‐association equilibria between the native enzyme and its protomers or systematic errors in the methods applied.To clarify this alternative the monodispersity of the homogeneous heart (H4) and muscle (M4) isoenzymes has been analysed comparing the mean values &#x004d;̄ from sedimentation, light scattering and osmosis.Considering all correction terms in the evaluations, the results in the total range of enzymic activity clearly establish the monodisperse tetramer as the native unit giving &#x004d;̄s,D=&#x004d;̄w=&#x004d;̄n= 142,000 ± 3,600. No anomalous dependence of the molecular data on protein concentration and temperature have been observed on calculating this figure.Dissociation into subunits requires high net charge or high ionic strength ás well as high concentrations of guanidine. HCI, or urea at high ionic strength. Under all conditions dissociation is accompanied by conformational changes thus restricting a more detailed insight into the native quaternary structure in solution. Regarding the interprotomer binding sites variation of the medium proves Coulombic attractions to prevail in the stabilization of the tetrameric state.
A Correlation Study between Myocardial Substrate Levels and the Blood Glucose Level in Normal, and in Acute and Chronic Alloxan‐Diabetic Rats in vivo
Authors: KRAUPP O. , ADLER‐KASTNER L. , NIESSNER H. , PLANK B. 
Published: March 2005

A correlation study has been carried out on data obtained by Kraupp et al. [1] for the myocardial levels of citrate, malate, 2‐oxoglutarate, acetoacetate, 3‐hydroxybutyrate, intracellular glucose (estimated), glucose 6‐phosphate, fructose 6‐phosphate, fructose 1,6‐diphosphate, pyruvate, lactate, glycogen, ATP and free fatty acids (FFA) in relation to the corresponding blood glucose level in I‐normal, II‐acute alloxan‐diabetic, and III‐chronic alloxan‐diabetic rats in vivo. A variation in blood glucose level within each group had been achieved by starvation of about 50% of the animals.In the normal rats there was a significant, negative correlation between the blood glucose level and the myocardial levels of citrate, malate and 2‐oxoglutarate, acetoacetate and 3‐hydroxybutyrate, and glycogen. A significant, positive correlation was obtained between the blood glucose level and the estimated intracellular glucose level and the fructose 1,6‐diphosphate/fructose 6‐phosphate ratio in the myocardium.In the diabetic rats, a generally reverse pattern was obtained. The acute alloxan‐diabetic rats showed a significant, positive correlation of the blood glucose level to the myocardial levels of citrate and malate (not 2‐oxoglutarate), acetoacetate and 3‐hydroxybutyrate, estimated intracellular glucose, hexose monophosphates, lactate, and the lactate/pyruvate ratio. The fructose 1,6‐diphosphate/fructose 6‐phosphate ratio was negatively correlated with the blood glucose level.In the chronic alloxan‐diabetic rats myocardial citrate, malate, estimated intracellular glucose, glucose 6‐phosphate and pyruvate were positively correlated with the blood glucose level. The negative correlation of the fructose 1,6‐diphosphate/fructose 6‐phosphate ratio to the blood glucose level became of even greater significance than in group II (acute diabetic) animals and a negative correlation in respect to the myocardial ATP level appeared. The myocardial ketone‐body levels were uniformly low and showed no correlation to the blood glucose level.In no group was there any correlation between cardiac FFA levels and the blood glucose level.In another series of experiments with fed and starved, acute alloxan‐diabetic rats, there was no correlation between the blood glucose level and the extracellular levels of FFA, ketone bodies, lactate or pyruvate, nor the 3‐hydroxybutyrate/acetoacetate and lactate/pyruvate ratios.A working theory is proposed for the correlation between certain myocardial substrate levels and the blood glucose level in diabetic rats.
Early Effects of Phytohaemagglutinin on Lymphocyte RNA Synthesis
Authors: KAY J. E. 
Published: March 2005

Phytohaemagglutinin was found to cause a rapid increase in the incorporation of [3H]‐uridine into lymphocyte RNA and an increase in the rate of incorporation of [14C]leucine into protein which began about 2 h after its addition.
During the first few hours after its addition phytohaemagglutinin stimulated the labelling of cytoplasmic RNA more than the labelling of nuclear or whole cell RNA.
The cytoplasmic RNA labelled during the first 2 h of incubation of lymphocytes with phytohaemagglutinin sedimented mainly at 4 S when analyzed by sucrose gradient centrifugation, but part of it could be distinguished from transfer RNA. Phytohaemagglutinin stimulated the labelling of both fractions of the labelled cytoplasmic RNA more than the labelling of whole cell RNA.
Labelled ribosomal RNA was first found in the cytoplasm after incubation of lymphocytes with [3H]uridine for 3 h. Incubation with phytohaemagglutinin markedly stimulated the labelling of this fraction of RNA.
Dosage des pyridine nucléotides oxydés et réduits dans le sang et les tissus animaux
Authors: CARTIER P. H. 
Published: March 2005

A fluorometric method is described for the estimation of di‐ and triphosphopyridine nucleotides (oxidized and reduced) in animal tissues.The continuous cycling reduction of DPN+ (or TPN+) in the presence of excess of ethanol and alcohol dehydrogenase (or glucose‐6‐phosphate and glucose‐6‐phosphate dehydrogenase) was coupled with non enzymatic reoxidation of pyridine nucleotides with phenazine methosulfate.Phenazine methosulfate reduced the non fluorescent compound, resazurine to the highly fluorescent compound, resorufine. The rate of production of resorufine is proportionnal to the concentration of pyridine nucleotides.The procedure is relatively simple and requires only two commercial enzyme preparations for complete determination. This method can be used for estimation of pyridine nucleotides in concentrations as low as 0.1 μM and quantities as small as 10 pmoles.The acid and alkaline extraction procedure was carried out essentially according to the method of Lowry. Conditions affecting the specificity, accuracy and reliability have been studied. During extraction, interfering material is formed, inhibiting the rate of reduction. A calibration procedure is described which overcomes this difficulty. Values for the concentrations of oxidized and reduced pyridine nucleotides in rat liver and in rat and human blood are presented and are in fairly good agreement with those of Lowry.