bluuebunny/crossref_metadata_2025_split huggingface dataset:
Relationship between Extracellular Amino Acids and Protein Synthesis in vitro in the Rat Pancreas
Authors: van Venrooij Walter J. , Poort Cees , Kramer Mebius F. , Jansen Marcel T. 
Published: March 2005

The uptake and incorporation in vitro of radioactive leucine and lysine in rat pancreas fragments have been studied.
The uptake and incorporation of both leucine and lysine were found to be influenced by the concentration of leucine in the medium.
Extracellular and intracellular free amino acids can be distinguished by washing the tissue fragments at 0 °C.
Though it takes some 20 min for each of both radioactive amino acids to reach maximum concentration in the intracellular soluble amino acid pool, their incorporation into protein becomes linear shortly after the start of incubation in a medium with labelled amino acids.
In chase‐type experiments the incorporation of each of both radioactive amino acids stops immediately when the labelled medium is replaced by unlabelled medium, though the free, intracellular amino‐acid radioactivity declines only slowly.
These results indicate that in the pancreas of the fasting rat in vitro the amino acid molecules incorporated into protein are derived from the cxtracellular pool. Other molecules enter and leave the intracellular amino acid pool but do not contribute to incorporation.
Rameau dégradatif commun des hexuronates chez Escherichia coli K12
Authors: Pouysséqur Jacques M. , Stoeber Françis R. 
Published: March 2005

After the recent investigation on the biochemical properties of the 2‐keto‐3‐deoxy‐glucono‐kinase and 2‐keto‐3‐deoxy‐6‐phospho‐gluconate aldolase in Escherichia coli K12, we report in this paper the induction mechanism of these two enzymes and the genetic control of a new enzymatic activity: the 2‐keto‐3‐deoxy‐gluconate transport system.The d‐glucuronate and d‐galacturonate raise the high basal levels of the base and aldolase on glycerol by a factor of 5 and 3–4, respectively, the kinase and aldolase being enzymes of the common degradative pathway of d‐glucuronate and d‐galacturonate. The differential rates of synthesis of these two enzymes are constant from the addition of the galacturonate. Under a variety of conditions (different inducers and substrates of growth) kinase and aldolase have been found to be synthesized non coordinately. The strongest decoordination is carried out by gluconate: this compound which is a good inducer of the aldolase, not only is unable to induce the kinase but also represses it. Moreover, the aldolase seems to be less sensitive to the catabolic repression than the kinase.By means of appropriate negative mutants we have established that the two hexuronates and two keto‐uronates do not induce directly kinase and aldolase, but by sequential conversion into 2‐keto‐3‐deoxy‐gluconate. Furthermore, the fact that the hexuronates induce the kinase in aldolase‐negative mutants (kdg A) and besides, “over” induce the aldolase in kinase negative mutants (kdg K) strongly suggests that 2‐keto‐3‐deoxy‐gluconate is a true inducer for both enzymes. In addition, the gluconate which still induces the aldolase in an edd strain suggests that, this compound and/or the 6‐phospho‐gluconate are/is also inducer(s) of this enzyme.Moreover, we have isolated spontaneous mutants able to utilize the 2‐keto‐3‐deoxy‐gluconate as a unique carbon source. Two classes have been identified. The mutants of these classes which arise at high frequency (10−5 to 10−6) are either simultaneously constitutive for a specific 2‐keto‐3‐deoxy‐gluconate transport system, kinase and aldolase (class kdg Rc)or only constitutive for the transport system (class kdg Pc). The 2‐keto‐3‐deoxy‐gluconate as exogenous substrate, which cannot induce its transport system in the wild type, behaves like a non‐inducer substrate. So the strains kdg Rc and kdg Pc are respectively i− and Oclac‐like mutants. These findings and the fact that we have found thermosensitive mutants mapping in the kdg R locus (34.5 min) and which are simultaneously derepressed for the permease, kinase and aldolase at 42°C but not at 28°C, strongly suggest that the synthesis of the three sequential enzymes degrading the 2‐keto‐3‐deoxy‐gluconate is negatively controlled by a common regulator gene product. The decoordinated syntheses of these three enzymes are in agreement with the scattering of the corresponding operons on the chromosome: transport system operon (76.5min), kinase operon (69 min), aldolase operon (34 min).
Thermodynamic and Kinetic Characterization of Electron‐Transfer Components in situ in Rhodopseudomonas spheroides and Rhodospirillum rubrum
Authors: Dutton P. Leslie , Jackson J. Barry 
Published: March 2005

The b‐ and c‐type cytochromes in chromatophores from photosynthetically grown Rhodospirillum rubrum and Rhodopseudomonas spheroides have been characterized in situ in terms of their oxidation‐reduction potential properties.In R. rubrum only one cytochrome c measured at 551—540 nm was resolved potentiometrically; the midpoint potential at pH 7.2, Em7.2, is +293 mV. Three cytochrome species measured at 562—540 nm were resolved; they had Em7.2 values + 170 mV, −5 mV and −105 mV and were tentatively identified with cytochrome b, cc′ and b respectively; they contributed to the over‐all absorbance change in the ratio 8:25:17, respectively.In Rps. spheroides the Em7.0 of cytochrome c is +295 mV. Three cytochromes tentatively considered to be of the b‐type have Em7.2 values of +155 mV, +50 mV and −90 mV; their approximate reduced minus oxidized maxima were determined to be 558—9 nm, 560 nm and 564 nm, respectively. The approximate relative absorbance ratio given at the reduced minus oxidized maxima was 8:21:14.
The rapid oxidation‐reduction reactions of reaction‐center bacteriochlorophyll, cytochrome c and cytochromes bin Rps. spheroides measured following a 20‐nsec laser flash or a 200‐μsec xenon flash have been studied in the absence and presence of antimycin A as a function of oxidation‐reduction potential. The results indicated that the reaction‐center bacteriochlorophyll (Em7.0+ 450 mV) serves to oxidize the cytochrome c with a midpoint potential of 295 mV, cytochrome c295, (half‐time t1/2↗100 μsec with a slow phase t1/2∼2 msec) which in turn can oxidize cytochrome b155t1/2 1—2 msec) in a reaction which is antimycin‐A‐sensitive. Cytochrome b50 becomes reduced following a flash with a t1/2 1–1.5 msec probably via the primary electron acceptor (Em7.0∼−20 mV) of the reaction‐center bacteriochlorophyll. Antimycin A stimulates the extent of cytochrome b50 reduction following a 200‐μsec xenon flash but has no detectable effect following a 20‐nsec laser flash.
A working model for electron transfer in Rps. spheroides based on the measured thermodynamic and kinetic properties is presented.
Fructose andd‐Glyceraldehyde Metabolism in the Isolated Perfused Pig Liver
Authors: Sestoft Leif , Tønnesen Klavs , Vallø Hansen Finn , Damgaard Stig E. 
Published: March 2005

The metabolism of fructose andd‐glyceraldehyde by the isolated perfused pig liver and kinetics of relevant pig‐liver enzymes were studied.Fructose elimination by the isolated perfused organ could be described by Michaelis kinetics with an apparentKmof 5.9 mM and a maximum elimination rate of 2.9 μmol × g−1× min−1. The Kmfor pig liver ketohexokinase was 0.5 mM and the maximum activity 1.9 U/g at 25°C.Fructose 1‐phosphate up to 6.3 mM was measured in biopsies taken at the end of the experiments.The reversible inhibition of the aldolase cleavage of fructose 1‐phosphate was demonstrated by renewed formation ofd‐glyceraldehyde after an initial high dose of fructose had been eliminated. During steady‐state fructose elimination the concentration ofd‐glyceraldehyde in the medium was below 0.5 mM.The elimination kinetics ofd‐glyceraldehyde did not follow simple Michaelis kinetics, and the maximum elimination rate could not be measured.d‐glyceraldehyde was metabolized to glycerol, glycerate, and (probablyviatriokinase) to lactate. Net glycerol formation took place only at concentrations ofd‐glyceraldehyde above 1 mM. Glycerate accumulated even at low concentrations ofd‐glyceraldehyde. The findings are in accordance with the kinetics of the appropriate enzymes measuredin vitro.More glucose than lactate was formed from fructose, while the reverse was true ford‐glyceraldehyde. Lactate, pyruvate, glucose, glycerol, glycerate,d‐glyceraldehyde, sorbitol, malate, citrate, glutainate, and ketone bodies accounted for less than half the amount of fructose metabolized. The concentration of sorbitol, malate, and citrate rose during fructose metabolism.Fructose causes a small transient fall in the lactate to pyruvate concentration ratio.The oxygen consumption was increased 30% by fructose.
The Mechanism of Adenovirus‐DNA Synthesis in Isolated Nuclei
Authors: van der Vliet Peter C. , Sussenbach John S. 
Published: March 2005

KB cells were productively infected with adenovirus type 5 and nuclei were isolated during the period of active viral DNA replication. The DNA synthesis in isolated nuclei was followed by studying the incorporation of deoxyribonucleoside triphosphates, including dBTP, which replaced dTTP completely.Sucrose gradient centrifugation of new DNA, synthesized during a 2‐h incubation, revealed the presence of three classes of viral DNA, one cosedimenting with mature viral DNA (31 S), one faster‐sedimenting class which contained replicating molecules and a slower‐sedimenting class which was not further investigated. Newly synthesized 31‐S DNA banded in CsCI within the range of light‐to‐hybrid DNA while less than 3% banded at higher densities. This suggests the absence of reinitiation of new rounds of DNA replication during the incubation period. The DNA synthesis was shown to proceed in a semiconservative fashion with a rate of at least 50% of the rate reported to occur in intact cells.By means of pulse labelling with [3H]thymidine of DNA in intact cells followed by incubation of nuclei in the presence of dBTP it was found that a large fraction of the DNA strands involved in replication in intact cells was covalently bound to DNA synthesized in the nuclear system.This indicates that DNA synthesis in isolated nuclei continues at the replication fork that existed in the intact cells. During this process replicating molecules can be completed. Infection with adenovirus type 5 containing [32P]DNA showed that parental DNA participated in the replication process. A covalent binding between parental and new DNA was not observed, neither in replicating DNA nor in the 31‐5 mature DNA. The latter observation showed the absence of extensive repair synthesis.
Oligonucleotide Conformations
Authors: Guschlbauer Wilhelm , Frič Ivo , Holý Antonin 
Published: March 2005

Guanylyl‐3′‐5′‐uridine (GpU) and seventeen GpU analogues modified mostly on the uridine moiety have been investigated by circular dichroism (CD). The following conclusions can be drawn from this investigation.

At room temperature and neutral pH GpU is very little, if at all stacked. A perpendicular structure with guanosine in syn and uridine with ϕCN∼+75°± 15°C stabilized by a hydrogen bond between the amino group of guanosine and the C‐4 keto group of uridine is proposed. In this structure C‐5 and C‐6 would be hidden by the guanosine base.
At low temperature or low pH stacking occurs. The structure is possibly the same under these conditions and may involve guanosine in syn and uridine in anti.
Substitution at C‐5 of uridine inhibits the formation of the neutral perpendicular structure. Seven C‐5 substituted GpU analogues all show CD spectra close to those of GpU at low temperature or low pH.
Substitution on C‐6 of uridine destabilizes the formation of all these structures.
Conformation of Lysozyme in Aqueous Solution
Authors: Hampe Oscar Geraldo 
Published: March 2005

The ionic strength, concentration, and temperature effects on the association‐dissociation properties of lysozyme in aqueous solution have been studied by gel chromatography. The following results were obtained:

Increasing ionic strength leads to an increase in inter‐conversion rate between the monomeric and associated forms. Between distilled water and 0.01 ionic strength, lysozyme shows a low velocity of interconversion, whereas between 0.01 and 0.2 rates are observed which are rapid as compared with the column fractionation time.
Analysis of concentration dependence indicates the formation of more highly associated forms than the dimer. The data can be fitted within experimental error by an indefinite association with the same equilibrium constant for the addition of each successive monomer.
The equilibrium ratio for the slow polymerization process measured at temperatures between 15° and 40°C yields the apparent thermodynamic quantities, ΔG°, ΔH0, and ΔS0 for the association reaction. This data is consistent with a hydrophobic interaction between lysozyme monomers and Sephadex gel matrix. It is suggested that the adsorption involves the tryptophan residues of the active centre. The enthalpy of the polymerization process is interpreted in terms of a conformational change of the same order of magnitude as on the bonding of N‐acetylglucosamine to the enzyme.
Regulation of a Catabolic Pathway
Authors: Vandecasteele Jean‐Paul , Hermann Monique 
Published: March 2005

The regulation of the pathway responsible for l‐lysine degradation in Pseudomonas putida has been studied. It has been shown that l‐lysine is the actual inducer of the two first enzymes of the pathway, lysine oxygenase and 5‐aminovaleramidase.
No important repression of lysine oxygenase by various carbon sources and by intermediates of the lysine degradation pathway has been found.
Activation of lysine oxygenase by its substrate, lysine, has been observed. A physiological function for this phenomenom is proposed.
Sodium and potassium ions are activators whereas magnesium ion, phosphate and phosphorylated compounds are inhibitors of lysine oxygenase. Citrate also is inhibitory.
The Use of Glucosamine as a Metabolic Probe in the Rat Diaphragm
Authors: Khac Lien Do , Eboué‐bonis Dominique , Chambaut Anne‐Marie , Clauser Hubert 
Published: March 2005

The action of insulin on [14C]glucosamine uptake and metabolism is analyzed in the isolated rat diaphragm. Various metabolites accumulating in the course of incubation are extracted, characterized and estimated by chromatographic, electrophoretic and colorimetric procedures.
Insulin greatly stimulates (up to three‐fold) the uptake and time‐dependent accumulation of metabolites derived from glucosamine. It is demonstrated that glycogen accounts but for a small part (less than 20%) of the accumulated material; the major part consists of glucosamine 6‐phosphate, the level of which is increased up to six times by insulin in the cell. Hence the hormone affects glucosamine metabolism already at the level of its first steps: transport and phosphorylation.
The use of d‐glucose and 3‐O‐methyl‐d‐glucose as competitive inhibitors of glucosamine metabolism shows that the mechanisms by which all three substrates are transported and by which two of them (glucose and glucosamine) are phosphorylated are essentially identical, both in the absence and in the presence of insulin.
The action of phlorizin as an inhibitor of sugar transport confirms this interpretation.
The results obtained are consistent with the hypothesis of an insulin‐stimulated, facilitated diffusion step of glucosamine and of a bottle‐neck reaction, which limits the deamination of glucosamine 6‐phosphate and leads to its accumulation in the tissue.
Purification and Partial Characterisation of Rat‐Liver Nuclear DNA Polymerase
Authors: Haines Michael E. , Wickremasinghe R. Gitendra , Johnston Irving E. 
Published: March 2005

A method is described for the preparation of DNA polymerase purified about 800‐fold from rat liver nuclei. The yield of enzyme is about 140–200 μg from 200 g liver.Sodium dodecylsulphate‐polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29000 ± 3%. Sephadex G‐100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42000.The enzyme prefers double‐stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside‐5′‐triphosphate in the assay.The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it.
Crystallization of Pure Species of Bacterial tRNA for X‐Ray Diffraction Studies
Authors: Brown Raymond S. , Clark Brian F. C. , Coulson Robert R. , Finch John T. , Klug Aaron , Rhodes Daniela 
Published: March 2005

Three pure species of bacterial tRNA have been crystallized by several different methods for X‐ray diffraction studies.
Extensive studies of tRNAfMet produced many crystal forms, seven of which are noted here. Crystallization conditions for both tRNATyr and tRNAPhe have been systematically investigated for a limited number of solvents. Several forms of tRNATyr can be produced in a controlled fashion.
The most favourable unit cells are P4122, a= 7.1 nm, c= 17.4 nm for tRNATyr and P3112, a= 9.3 nm, c= 7.8 nm, for tRNAphe. Initial X‐ray still photographs of tRNATyr and tRNAPhe show some reflections out to 0.4 nm but precession photographs show a rapid fall off in intensity beyond 0.7 nm.
Evidence for Induced Fit of a Pseudo‐Substrate of Aspartate Aminotransferase
Authors: John Robert A. , Tudball Norman 
Published: March 2005

Cytoplasmic aspartate aminotransferase from pig heart catalyses β‐elimination of l‐serine‐o‐sulphate and β‐chloro‐l‐alanine. The rates of these reactions have been compared with the rates of the same reactions catalysed by free pyridoxal 5′‐phosphate and CuCl2.
The effect of formate on the two enzyme‐catalysed reactions has been studied and it has been shown that this anion competitively inhibits the serine sulphate reaction and activates the chloroalanine reaction by increasing kcat considerably.
The β‐elimination of both these compounds is accompanied by progressive inactivation of the enzyme but whereas formate decreases the rate of inactivation by serine sulphate it increases the rate of inactivation by chloroalanine.
It is proposed that formate binds to the active site of the enzyme in a position normally occupied by the β‐carboxyl group of aspartate, the carboxyl group of glutamate or the sulphate group of serine sulphate and that binding of the appropriate anion induces a change in the active site which accounts simultaneously for increased catalytic activity and increased inactivation.
Incorrect Aminoacylations Catalysed by the Phenylalanyl‐ and Valyl‐tRNA Synthetases from Yeast
Authors: Kern Daniel , Giegé Richard , Ebel Jean‐Pierre 
Published: March 2005

It has been found that purified phenylalanyl‐ and valyl‐tRNA synthetases from yeast catalyse the mischarging of numerous heterologous Escherichia coli and even of homologous yeast tRNAs when special aminoacylation conditions are used. For instance E. coli tRNAAla, tRNAVal, tRNALys and yeast tRNAAla, tRNAVal can be incorrectly aminoacylated by yeast phenylalanyl‐tRNA synthetase, whereas E. coli tRNAAla, tRNAMet, tRNAIle, tRNAThr and yeast tRNAAla, tRNAPhe can be mischarged by yeast valyl‐tRNA synthetase. The production of errors is highly dependant on the experimental conditions used, as their number and their level is increased when the Mg2+/ATP ratio or the enzyme concentration in the medium is increased or when the reactions are performed in the presence of dimethylsulfoxide. In the case of phenylalanyl‐tRNA synthetase, conditions can be found where practically all E. coli tRNAs are wrongly aminoacylated, although at different levels. On the other hand, in the case of homologous systems some errors can only be detected when purified tRNAs are used, thus suggesting, when total tRNA is used, a competition between the cognate and non‐cognate tRNAs which minimises the mischarging.The comparison of the sequences of the cognate and non cognate tRNAs which are aminoacylated either by the yeast phenylalanyl‐ or valyl‐tRNA synthetase led us to ascribe some importance to several regions inside of these tRNAs, for instance (a) the dihydrouracil arm, (b) the terminal part of the amino‐acid acceptor stem and (c) the extra‐loop. These regions should be essentially necessary for the establisment of the correct tri‐dimensional conformation necessary for the recognition by the aminoacyl‐tRNA synthetases.
Protein Synthesis in Mitochondria of Euglena gracilis
Authors: Kislev Naomi , Eisenstadt Jerome M. 
Published: March 2005

Mitochondria purified from streptomycin‐bleached mutant SB3 (Krawiec and Eisenstadt, 1970) of Euglena gracilis Klebs strain z, were found to be active in incorporation in vitro of labeled amino acids into proteins. Chloramphenicol and puromycin were most effective inhibitors for the system, while cycloheximide has very little effect. Incorporation of the amino acids was resistant to added DNAase and RNAase.tRNA and tRNA synthetases obtained from mitochondrial fractions were active in vitro and were found to be different from those obtained from chloroplasts of green Euglena gracilis for the amino acids tested.
Adenylate Cyclase and Insulin Action
Authors: Hepp K. Dieter 
Published: March 2005

Activation of adenylate cyclase in a particle preparation from mouse liver was studied after previous exposure to synthetic glucagon and NaF, and following extensive washing of the preparation. Activation by glucagon was maintained, but fully abolished when GTP was preincubated with the hormone. ATP reduced the effect of glucagon and NaF.
The effect of submaximal concentrations of glucagon was significantly inhibited in the presence of physiological amounts of insulin (50–100 μU/ml). Increasing the concentration of insulin led to a disappearance of this effect. Whereas reduction of the basal adenylate cyclase activity was observed, no effect was obtained under maximal stimulation by NaF.
Nonsuppressible insulin‐like material, a polypeptide with a molecular weight of approx. 7000, which mimics insulin in a number of metabolic effects, was also found to inhibit the basal as well as the glucagon‐stimulated adenylate cyclase. In contrast to insulin, no paradoxical decrease was seen with higher concentrations. The effects of high concentrations of NaF were not inhibited by insulin‐like material. After previous incubation with insulin‐like material its effect was maintained in the subsequent assay suggesting that the substance binds to liver membranes. In mouse myocardium, insulin‐like material caused no reduction in norepinephrine‐stimulated adenylate cyclase activity.
Cyclic‐AMP phosphodiesterase activity, as measured with 1.13 μM substrate, was not influenced by insulin or insulin‐like material. It is unlikely that increased breakdown of cyclic AMP during the adenylate cyclase reaction is responsible for the observed effect of insulin or insulin‐like material.
Diabetes, induced by streptozotocin, led to a marked increase in the sensitivity of the adenylate cyclase system to maximal glucagon stimulation. In contrast, the sensitivity of the system to NaF remained unchanged. Insulin treatment of diabetic animals in vivo led to a reversal of the glucagon effect towards the values in the control group.

It is suggested that both insulin and insulin‐like material directly affect the adenylate cyclase system by interference with the transmission of the glucagon signal between the receptor and the catalytic enzyme unit. This effect may be of fundamental physiological significance in the mechanism of action of insulin.