bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectral Studies of the Interaction of Bovine α‐Lactalbumin and Egg‐White Lysozyme with 2‐p‐Toluidinylnaphthalene‐6‐sulfonate
Authors: Barel André O. , Turneer Mireille , Dolmans Marcel
Published: March 2005

The extrinsic fluorescence of a bound dye, 2‐p‐toluidinylnaphthalene‐6‐sulfonate was used in order to study and to compare the hydrophobic sites on the surface of bovine α‐lactalbumin and egg‐white lysozyme.

Both α‐lactalbumin and lysozyme bind one mol toluidinylnaphthalenesulfonate per mol protein, as determined fluorimetrically.

The dissociation constants of the protein · dye complex for the two proteins are similar (0.3 to 0.5 mM), indicating a moderate binding of the dye to lysozyme and α‐lactalbumin.

Considering the fluorescence of the protein · dye complex and the position of the maximum in the emission spectrum, it was found that the binding site on α‐lactalbumin was more hydrophobic than lysozyme.

Binding experiments of lysozyme with toluidinylnaphthalenesulfonate in the presence of a competitive inhibitor (N‐acetyl‐d‐glucosamine) and measurements of bacteriolytic activity in the presence of this dye, suggest that the binding site on lysozyme did not significantly overlap with the area involved in the catalytic site.

The intensity of fluorescence of the bound dye in α‐lactalbumin is diminished to a small extent by the presence of various sugars (glucose, lactose and galactose). The dissociation constant of the α‐lactalbumin · dye complex is identical in the presence or in the absence of these sugars.
Calorimetric Titration of Lysozyme
Authors: Bjurulf Carin
Published: March 2005

A method is described for enthalpy titration of proteins, using a flow microcalorimeter in combination with a device for continuous pH measurements.

The calorimetric titration of lysozyme between pH 1.5 and 12 has been carried out as well as the corresponding potentiometric titration. Enthalpy values for the titrated groups are estimated and discussed in comparison with reported data for free amino acids.
Synthesis of Lens Protein in vitro Role of Methionyl‐tRNAs in the Synthesis of Calf‐Lens α‐Crystallin
Authors: Strous Ger J. A. M. , Berns Ton J. M. , van Westernen Hanske , Bloemendal Hans
Published: March 2005

The main polypeptide chain of the lens protein α‐crystallin (αA2) is N‐terminally acetylated and contains two methionine residues. The chain is initiated by the incorporation of a methionine residue exclusively donated by methionyl‐tRNArMet.

This N‐terminal methionine is not removed during polypeptide chain synthesis. The internal methionine residue is donated exclusively by one of the methionyl‐tRNAMet species. When lens messenger RNA coding for the αrA2 chain is translated in a reticulocyte cell‐free system, the newly synthesized polypeptide chain carries an acetylated methionine in the N‐terminal position.
Pyruvate Metabolism in Mitochondria from Rat Liver
Authors: Stucki Jörg W. , Walter Paul
Published: March 2005

Rat liver mitochondria were incubated in the presence of [2‐14C]pyruvate, HCO3−, ATP and phosphate. From the measured amounts and the specific radioactivities of the products formed, metabolic fluxes through individual enzymatic reactions of pyruvate metabolism including the citric acid cycle were calculated. These fluxes and the formation of the products were simulated with a computer‐model which involved 51 reactions and 39 metabolites.

Computer‐simulations and calculations of the experimental results revealed the existence of energy‐dissipating processes. About one third of the energy‐dissipation was inhibited by atractylate. It is concluded that this atractylate‐sensitive energy dissipation was due to some extramitochondrial ATPase and to the maintenance of an asymmetric distribution of ATP and ADP across the inner membrane. The atractylate‐insensitive part of energy dissipation is probably related to state 4 respiration of isolated mitochondria.

Based on the specific radioactivities of malate and citrate it was concluded that the pools of malate and oxaloacetate within the matrix cannot be fully equilibrated. It is proposed that the transport of malate out of the mitochondria restricts the passage of this metabolite through the citric acid cycle.
Study of the Position of NAD and Its Effect on the Conformation of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase by Small‐Angle X‐Ray Scattering
Authors: Simon István
Published: March 2005

The structural changes of mammalian skeletal muscle d‐glyceraldehyde‐3‐phosphate dehydrogenase induced by the binding of the coenzyme, NAD, were studied by small‐angle X‐ray scattering with the double‐monitor technique.

As shown by our difference method the radius of gyration of the apoenzyme decreases by 0.08 nm on the binding of four molecules of NAD. The decrease in the radius of gyration is not a monotonous function of saturation with NAD.

From the analysis of our data it follows that, in the order of binding, the first and third NADs are positioned at a distance of about 2 nm from the centre of gravity of the molecule, whereas the second and fourth NADs are at a distance of about 4 nm.

As indicated by the change in the radius of gyration the protomers of the tetrameric molecule are dislocated by about 0.1–0.2 nm (i.e. 1–4°) relative to each other.
RNA Synthesis in Isolated Rabbit‐Liver Nuclei
Authors: Smith Kelvin E. , Arnstein Henry R. V.
Published: March 2005

At 5 mm Mg2+, isolated rabbit liver nuclei incorporated nucleoside triphosphate precursors into RNA. The incorporation was stimulated by low concentrations of monovalent cations. These nuclei also rapidly incorporated the active methyl group of S‐adenosyl‐l‐methionine into acid‐insoluble material. The majority of the label was shown to be in the nuclear protein fraction and not the RNA. In agreement with this finding, it was shown that the methylation reaction was not inhibited by actinomycin D whereas RNA synthesis was inhibited. We present evidence showing that a very small percentage of the pulse‐labelled RNA was methylated in vitro. Finally, the nature of the pulse‐labelled RNA was examined using the techniques of sucrose gradient density centrifugation and base‐ratio analysis.
La d‐altronate: NAD‐oxydoréductase d' Escherichia coli K12
Authors: Portalier Raymond C.
Published: March 2005

Kinetic studies were carried out in order to investigate the enzymic mechanism of Escherichia coli K12 d‐altronate: NAD‐oxydoreductaso.

Initial velocity patterns (reciprocal plots) of the forward and reverse reactions are intersecting, referring to a sequential mechanism. The following numerical values of the Michaelis and dissociation constants are extracted graphically from secondary plots (slopes and intercepts of the reciprocal plots versus the reciprocal concentrations of the non‐varied substrate): Kia= 5.8 μM, Ka= 85 μM and Kb= 670 μM for the forward reaction; Kia, = 156 μM, Ka= 53 μM and Kb= 75 μM for the reverse reaction.

At pH 6.3, in phosphate buffer, product‐inhibition patterns of the forward reaction suggest an ordered bi‐bi mechanism for the altronate oxydoreductase with NADH as the first substrate to add to the enzyme. The only competitive inhibition observed is when NAD+ is the product inhibitor and NADH the variable substrate, all others inhibitions being non competitive.

At pH 8.9, in glycylglycine buffer, product inhibition patterns of the reverse reaction also revealed an ordered bi‐bi mechanism with altronate as the first substrate to add to the enzyme. The only competitive inhibition observed is when tagaturonate is the product inhibitor and altronate the variable substrate, all others inhibitions being non competitive.

Dead‐end product inhibition studies with fructuronate, mannonate and ATP, and binding studies in relation to the protection of the enzyme against thermal inactivation, are consistent with an ordered bi‐bi enzymic mechanism for the altronate oxydoreductase.

The occurrence of two possible enzymic forms of the altronate oxydoreductase according to the physical properties of the reaction medium is suggested.
Arginyl‐Transfer Ribonucleic‐Acid Synthetase from Bacillus stearothermophilus
Authors: Parfaitand Raymond , Grosjean Henri
Published: March 2005

Arginyl‐tRNA synthetase from Bacillus stearothermophilus (NCA 1518) has been purified approximately 450‐fold to 90%, homogeneity. The enzyme has a molecular weight of 78000 as estimated by disc electrophoresis in denaturing conditions (dodecylsulfate). No subunits were revealed in these conditions. The Km values for arginine, ATP and tRNA have been determined. Like the arginyl‐tRNA synthetase from other organisms, this one from B. stearothermophilus requires tRNA to catalyse an arginine‐dependent ATP/[32P]PPi exchange reaction. Periodatetreated tRNA does not induce this exchange reaction. Aminoacylation is not completely inhibited by 2 mM pyrophosphate, indicating that the exchange reaction may be explained by the reversal of the overall aminioacylation reaction.

Different methods tested failed to reveal any arginyl‐adenylate intermediary. By gel filtration, however, enzyme‐bound arginine could be isolated from a system containing all the substrates of the enzyme.

The binding of arginine and ATP to arginyl‐tRNA synthetase has been studied by equilibrium partition and shows that arginine is bound only after the binding of tRNA and ATP.

The binding of ATP does not depend on the presence of other substrates in the reaction mixture. One binding site for arginine and one for ATP are found per molecule of active enzyme. Periodate‐oxidized tRNA and phenoxyacetylarginyl‐tRNA did not induce the binding of arginine to the enzyme. Intact tRNA from other species which could be aminoacylated by arginyl‐tRNA synthetase from B. stearothermophilus, also induced the binding of arginine to the enzyme. Nucleotides other than ATP were not effective in the binding of arginine.

It is concluded that the mechanism of action of this enzyme is a concerted one, as proposed by Loftfield and Eigner, in which arginine is bound after tRNA and ATP.
Effect of Fructose and Glyceraldehyde on Ethanol Metabolism in Human Liver and in Rat Liver
Authors: Thieden Herluf I. D. , Grunnet Niels , Damgaard Stig E. , Sestoft Leif
Published: March 2005

The basic kinetic parameters, V and Km, have been determined for liver enzymes involved in the metabolism of fructose and ethanol in rats and man. Values, previously not reported, or which deviate significantly from those reported in the literature are as follows: The maximal activity of aldehyde dehydrogenase from human liver with acetaldehyde as substrate was determined as 43 μmol × min−1× g wet wt−1. The activity of NADP‐dependent alcohol dehydrogenase with ethanol as substrate both in rat liver and in human liver was very low. The presence of glycerate kinase (12 μmol × min−1× g wet wt−1) in human liver has been established. The Km‐value of human‐liver alcohol dehydrogenase (NAD) for d‐glyceraldehyde was determined as 80–90 mM compared to 8–10 mM for the rat liver enzyme. The NADP‐dependent alcohol dehydrogenase from human liver had a Km‐value for d‐glyceraldehyde of 2.5–3.3 mM. Glycerate kinase from human and rat liver had Km‐values for d‐glyceraldehyde of 2.5–3.0 mM and 0.03 mM, respectively.

In slices of human liver the increase in ethanol‐oxidation rate caused by 11 mM fructose (‘the fructose effect’) or by 2 mM d‐glyceraldehyde was 76% and 56%, respectively.

In rat‐liver slices the effect of D‐glyceraldehyde upon ethanol metabolism was inhibited 100% by rotenone. In the same experiments rotenone caused a 5001, inhibition of the basic ethanol oxidation rate and of the oxygen consumption.

10 mM pyruvate, in experiments with liver slices from fasted rats, caused a 90%, increase in the ethanol oxidation rate in the absence of CO2, but only a 20%, increase in the presence of CO2 CO2 in itself caused a 70%, increase in the unstimulated oxidation of ethanol.

Kinetic considerations and results reported in this paper held together with results from other laboratories lead to the conclusion that the theories previously proposed for the effect of fructose, d‐glyceraldehyde or pyruvate upon ethanol metabolism are irreconcilable with the experimental results. A new hypothesis for the ‘fructose effect’ is proposed.

Malate dehydrogenase, malic enzyme, pyruvate carboxylase and transfer of oxaloacetate from the mitochondrial to the extramitochondrial compartment constitute a mechanism by which transfer of hydrogen from NADH to NADP may take place, thus increasing the rate of removal of NADH from the cytoplasm.
Arginase Induction in Aspergillus nidulans
Authors: Cybis Jan , Weglenski Piotr
Published: March 2005

The induction of arginase in the fungus Aspergillus nidulans depends on the synthesis of messenger and its subsequent translation, as is shown by selective inhibition of the two processes by proflavine and actidione. In mycelium grown at 30°C messenger synthesis starts with no measurable delay but the enzyme appears 6 min after arginine addition. The stability of the enzyme is accompanied by the lability of its messenger, the coding capacity of which decays with the half‐life of 2.7 min. The synthesis of messenger can be separated from its translation by including actidione (5 μg/ml) in an arginine‐supplemented culture. After the withdrawal of inducer and inhibitor the expression of arginase messenger takes place. The 4.5‐fold increased stability of messenger observed under conditions of blocked translation may be responsible for its accumulation in presence of actidione.
The Modification of the Reconstituted Sarcoplasmic ATPase by Monovalent Cations
Authors: The Rudy , Hasselbach Wilhelm
Published: March 2005

The effects of monovalent cations on the calcium‐dependent ATPase activity of lipid‐depleted and reconstituted sarcoplasmic membranes have been investigated.

The calcium‐dependent ATPase activity of sarcoplasmic membranes whose lipid components have been modified are significantly more sensitive to activating and/or inhibitory effects of monovalent cations than that of native sarcoplasmic membranes.

Activating and inhibitory effects of the applied monovalent ions depend on the concentration of ionized calcium in the medium. In dilute calcium media ([Caz+] = 0.8 μM) modified ATPase preparations are always inhibited by high concentrations (>0.2M) of monovalent salts. Conversely, in concentrated calcium media ([Ca2+] = 0.18 mM) modified ATPase preparations are in most cases activated by increasing (>0.2 M) concentrations of monovalent salts.

Activating effects of the monovalent ions decrease and specific inhibitory effects increase in the order K+, Ria+, Rb+, Cs+, Li+. This order corresponds to that found if the ions are arranged according to the increasing distance between potassium and the respective ion on the hydration enthalpy scale.

The results have been discussed in relation to a specific competition between calcium ions bound to the inhibitory side at the inner surface of the sarcoplasmic membranes and the applied monovalent ions.
The Subunit Structure of the Pyruvate‐Dehydrogenase Complex from Escherichia coli K‐12
Authors: Vogel Otto , Hoehn Barbara , Henning Ulf
Published: March 2005

It is shown that very likely the Escherichia coli K‐12 pyruvate dehydrogenase complex is not a unique entity in that the amount of pyruvate dehydrogenase component it contains can vary. A complex can be obtained from which “excess” pyruvate dehydrogenase component is removed and this complex, called core complex, has an entirely reproducible polypeptide chain composition. The core complex has a molecular weight of 3750000 ± 200000.

In the core complex the dihydrolipoamide transacetylase component can consist of a polypeptide chain with a molecular weight of 80000 or of a number of fragments of this chain. Although fragmentation of the transacetylase need not influence the catalytic activity of the core complex it is operationally defined as that multienzyme complex which contains the transacetylase subunit with Mr= 80000.
Triiodothyronine Action on RNA Synthesis in Rat‐Liver Mitochondria
Authors: Gadaleta Maria N. , Barletta Antonio , Caldarazzo Maria , de Leo Teodoro , Saccone Cecilia
Published: March 2005

Chronic treatment of thyroidectomized rats with physiological doses of triiodothyronine leads to a significant increase in the incorporation of [3H]UTP into RNA by isolated rat liver mitochondria after the 4th day of treatment.

The increased activity of mitochondrial RNA polymerase is followed by a net increase in the RNA content of organelles which can be observed after the 5th day of chronic treatment.

Single injection of triiodothyronine at a dose of 250 μg/kg body wt also causes great stimulation of mitochondrial RNA synthesis which increases three‐fold after 40 h.

At the same DNA/RNA ratio, RNA labelled in vitro with [3H]UTP from liver mitochondria of thyroidectomized animals hybridizes with mitochondrial DNA less efficiently than the newly synthesized RNA from liver mitochondria of animals treated for 10 days with 5 μg/100 g body wt of the hormone.

Hybridization‐competition experiments with unlabelled RNA extracted from mitochondria of treated and untreated animals seem to suggest that a change in the transcription of mitochondrial DNA under the influence of the hormone occurs also in vivo.
Characterization of the Native and Denatured Conformations of tRNASerand tRNAPhefrom Yeast
Authors: Streeck Rolf E. , Zachau Hans G.
Published: March 2005

Previous work on the partial digestion of the native and denatured forms of tRNASerand tRNAPhefrom yeast with T1‐RNAase was extended. Detailed studies with pancreatic and T2‐RNAase and a few experiments with sheep kidney nuclease and acid RNAase from hog spleen revealed characteristically different fragmentation patterns for the native and denatured forms of the tRNAs. Apparently it is mainly the miniloop and dihydrouridine regions which are involved in the conformational changes responsible for denaturation.

The rate constants, activation enthalpies, and activation entropies of the renaturation process were calculated from amino acid acceptance data.

When the changes in hyperchromicity at 260 nm of the denatured tRNAs were followed as a function of time, a process was detected which in tRNAPheis 5–10 times and in tRNAser10–20 times faster than the renaturation as measured by acceptor activity. The whole net change in absorption, possibly representing the total net change in base stacking, occurs during the fast, process. The activation enthalpies and activation entropies are different for the fast and the over‐all processes.
Properties and Immunochemical Reactivities of Native and Modified Human‐Serum Albumin
Authors: Jacobsen Christian , Funding Lars , Møller Niels Peter H. , Steensgaard Jens
Published: March 2005

A series of chemically modified derivatives of human‐serum albumin has been produced in soluble monomeric form by modification of lysine, tryptophan, tyrosine, histidine, and acidic amino‐acid residues. Optical rotatory dispersion studies suggest that human‐serum albumin has a secondary structure which is resistant to many chemical side‐chain modifications. Sephadex‐gel‐filtration data and analytical ultracentrifugations indicate that the tertiary structure of human‐serum albumin is less resistant to chemical modification than the secondary structure. The combined data suggest that the secondary and tertiary structures to some extent are mutually independent. Lysine residues are found to be of major importance in the maintenance of the native tertiary structure.

Chemically modified derivatives of human serum albumin are found to have decreased abilities to combine with the corresponding rabbit antibodies by a modified Farr technique. By immunoelectrophoresis and immunodiffusion a 96% acetylation was required to destroy complete immunogenic determinants. Lysine residues were found to be of major importance to the immunochemical reactivity of human‐serum albumin, while tryptophan, acidic amino acids and tyrosine residues play a minor role. Histidine residues seem to be unimportant to the immunochemical reactivity,

Comparisons between immunochemical reactivities and structural parameters of native and modified albumins showed in particular a distinct relationship between the immunochemical reactivities and the Stokes‐radius values as determined by Sephadex‐gel filtration.
Inhibition of the Assembly of Cytochrome Oxidase in Neurospora crassa by Chloramphenicol
Authors: Sebald Walter , Weiss Hanns , Jackl Gerhard
Published: March 2005

Cytochrome oxidase was prepared from Neurospora crassa by chromatography on oleyl poly‐methacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation of labelled amino acids into the single polypeptides was investigated after a pulse labelling in the absence and presence of chloramphenicol, and after washing out the inhibitor.

Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90–95% while labelling of the whole membrane protein was inhibited only 30%. After washing out the inhibitor and further growth of the cells, the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a small increase in radioactivity. It is concluded that the four small‐sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol.
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.
An Unusual Group of Lysine‐Rich Histones from Gonads of a Sea Cucumber, Holothuria tubulosa
Authors: Phelan James J. , Subirana Juan A. , Cole R. David
Published: March 2005

Gonads of the male Holothuria tubulosa contain two families of lysine‐rich histones. One of these families resembles the lysine‐rich histones found in somatic tissues of higher organisms (e.g. calf and rabbit). The other family, which may be restricted to the male gonad, is recognizably related to the first family and yet is quite distinct. About 35% of the tryptic peptides differ between these families. These findings support the notion that a broad spectrum of structural variation may exist in lysine‐rich histones, perhaps even merging into structures of the slightly lysine‐rich class.
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.
Alkylation of Phosphates and Stability of Phosphate Triesters in DNA
Authors: Bannon Pierre , Verly Walter
Published: March 2005

A method is presented to measure the alkylation of phosphates in DNA after a treatment with an alkylating agent. Using this method, we have shown that phosphate alkylation represents 15% of total alkylation when DNA is alkylated with ethyl methanesulfonate and only 1% of total alkylation when DNA is alkylated with methyl methanesulfonate. Experiments are also presented which show that phosphate triesters resulting from the alkylation of DNA by ethyl methanesulfonate are very stable, most of them remaining intact after heating at 100°C for 90 min at pH 7.0.
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.
On the Presence of a Non‐Trimethylated iso‐1 Cytochrome c in a Wild‐Type Strain of Saccharomyces cerevisiae
Authors: Foucher Marcelle , Verdière Jacqueline , Lederer Florence , Slonimski Piotr P.
Published: March 2005

Ion‐exchange chromatography of the total cytochrome c extracted from a wild‐type laboratory strain of Saccharomyces cerevisiae shows the presence of two minor cytochrome c peaks, besides the already known iso‐1 and iso‐2 cytochromes c. Their amino acid composition is very similar to that of iso‐1, but one of them lacks the ɛ‐N‐trimethylated lysine residue characteristic of yeast cytochromes c, thus excluding, at least for this peak, an artifactual origin.
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.