bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetics of the Interaction between Pig‐Plasma Benzylamine Oxidase and Various Monoamines
Authors: LINDSTRÖM Anders , OLSSON Bengt , PETTERSSON Gösta , SZYMANSKA Jadwiga 
Published: March 2005

The kinetics of interaction between pig‐plasma benzylamine oxidase and a series of amine substrates have been investigated by conventional initial‐velocity studies, and by determination of transient rates of aldehyde formation and reduction of the enzymatic 470‐nm chromophore using stopped‐flow techniques. Kinetic parameters for the various processes and different substrates are reported.The results obtained provide evidence that reduction of the enzyme by different monoamines leads to the formation of a common reduced enzymatic intermediate, reoxidation of which appears to be rate‐limiting in all the reactions examined. Km‐values for the different substrates are inversely proportional to second‐order rate constants for the corresponding reduction process, and exhibit a striking correlation to the base strength of the respective amines. The latter observation may suggest that benzylamine oxidase acts exclusively on the non‐protonated form of amine substrates.
Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety
Authors: STÉVENIN James , JACOB Monique 
Published: March 2005

Previous results on RNA‐labelled nuclear particles were compatible with a folded ribonucleo‐protein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vivo; they were prepared by sonication of nuclei.1. Proteins were released progressively from the particles (ϱ= 1.39 g/ml) as shown by the accumulation of material of ϱ= 1.31–1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high‐density ribonucleoproteins were formed.2. Proteins released from the particles at various NaCl concentrations (up to 2 M) sedimented between 0 and 15 S. 30‐S aggregates corresponding to “informofers” were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCl.3. Large protein aggregates contaminated the smallest particles (up to 50–60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein‐labelled fractions.4. About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0–15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross‐linking. A 30‐S protein peak was not detected.5. The material released from the particles by ribonuclease banded at ϱ= 1.35 g/ml, a higher density than that of free proteins (ϱ= 1.31 g/ml). Treatment with 1 M NaCl after ribonuclease digestion released free proteins, some of which were phosphoproteins (ϱ= 1.31 g/ml) and ribonucleoproteins of higher density. It is suggested that these complexes contain ribonuclease‐resistant RNA.These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30‐S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins.
Transcription of Supercoiled Mitochondrial DNA by Bacterial RNA Polymerase
Authors: SCHMITT Henri , BECKMANN Jacques S. , LITTAUER Uriel Z. 
Published: March 2005

Mitochondrial DNA isolated from cysts of Artemia salina (brine shrimp) was found to have a closed‐circular structure 5.1 μm long and a buoyant density of 1.697 g/cm3. Escherichia coli RNA polymerase can transcribe this DNA. RNA synthesis is greatly stimulated by the σ subunit and is influenced by the presence of KCl. After formation of an initial complex between the enzyme and the mitochondrial DNA only a limited synthesis of RNA took place in the presence of rifampicin.The products obtained with holo enzyme and core enzyme in the presence of excess nucleoside triphosphates have a mean molecular weight of 1.5 × 106 and greater than 3 × 106 respectively, as determined by acrylamide‐agarose gel electrophoresis. Addition of the termination factor ϱ to the reaction mixture reduced the molecular weight of the products to 1.0 × 106 at a KCl concentration of 0.05 M but not at 0.1 M KCl.Under limiting UTP concentrations, a few discrete RNA bands of shorter size (about 8 × 105, 105 and 3 × 104 mol. wt) are synthesized. Addition of ϱ factor to the reaction mixture caused the disappearance of the higher‐molecular‐weight band (8 × 105) while the other RNA bands remained.Little self‐annealing was observed upon heating the [3H]RNA synthesized in vitro at 70 °C. On the other hand, when the [3H]RNA was heated in the presence of unlabeled mitochondrial rRNA, about 54% of the labeled product was resistant to degradation by a mixture of T1 and pancreatic RNase. In hybridization‐competition experiments, annealing of the [3H]RNA synthesized in vitro was reduced by 65% upon addition of unlabeled mitochondrial rRNA. RNA‐RNA annealing is thought to be the main cause for this competition reaction since similar results were obtained by simultaneous or step‐wise hybridization procedures. It is concluded that transcription in vitro of closed circular‐mitochondrial DNA from Artemia by E. coli RNA polymerase proceeds mainly on the anti‐rRNA strand.
Phosphorylation of Vinblastine‐Isolated Microtubules from Chick‐Embryonic Muscles
Authors: PIRAS Marta M. , PIRAS Romano 
Published: March 2005

Microtubule protein isolated from muscles of 11‐day‐old chick embryos migrates in gel electrophoresis with sodium dodecylsulphate–5% polyacrylamide as two closely associated bands of molecular weight 55000, which account for more than 90% total protein. Incubation of the micro‐tubular protein with [γ‐32P]ATP and Mg2+ results in a rapid incorporation of radioactivity into the 5% trichloroacetic acid precipitable fraction, without addition of any exogenous protein phosphokinase. The radioactivity can be solubilized by proteolytic enzymes and by alkali, and it migrates together with the faster moving band on gel electrophoresis. Elution of the tubulin band followed by acid hydrolysis gives rise to [32P]phosphorylserine. The protein phosphokinase activity associated to the microtubules is not stimulated by cyclic AMP or cyclic GMP and it can act on exogenous substrates, like casein, histone and phosphovitin. It emerges upon nitration through Bio‐gel P‐300 at a position of molecular weight 54000, separated from the bulk of the 32P radioactivity, which coincides with [3H]colchicine binding (molecular weight 120000). Phosphorylation by ATP (Km= 8 μM) requires Mg2+ or Mn2+. GTP can replace ATP in the phosphorylation reaction. Casein phosphorylation by ATP or GTP is affected only slightly by NaCl and vinblastine, but that of tubulin is affected to different extents, depending on the nucleoside triphosphate used.The micro tubular protein isolated from: (a) HeLa cells cultured in the presence of 32Pi; (b) muscle and brain slices of chick embryos incubated with 32Pi, and (c) muscles and brains of chick embryos injected with 32Pi, is labelled in all instances with 32P. The radioactivity migrates on gel electrophoresis like tubulin and gives rise upon elution and acid hydrolysis to [32P]phosphoserine. The results in vitro as well as those obtained in intact cells are discussed in connection with the possible role of phosphorylation as a control mechanism of microtubular function.
Transfer of Deuterium from [1‐2H2]Ethanol to Krebs Cycle and Related Acids of Rat Liver in vivo
Authors: CRONHOLM Tomas , MATERN Heidrun , MATERN Siegfried , SJÖVALL Jan 
Published: March 2005

The utilization in intermediary metabolism of hydrogens derived from C‐1 in ethanol was studied with the aid of [1‐2H2]ethanol. A gas chromatographic‐mass spectrometric method was developed and used to measure the deuterium content in individual positions of lactate, malate, 3‐hydroxybutyrate, citrate, succinate and fumarate isolated from freeze‐clamped livers of rats metabolizing [1‐2H2]ethanol (95 atoms%2H excess) at a maximal rate.A steady state of 2H‐labelling was reached within a few minutes. 3‐Hydroxybutyrate contained only 1–2 atoms% of deuterium which was interpreted to indicate that intramitochondrial NADH was essentially unlabelled due to exchange with protons in the NADH dehydrogenase reaction. A low labelling of lactate, 17 atoms%, indicated that the exchange of hydrogens in cytosolic NADH with unlabelled substrates and water was 4 times higher than the rate of ethanol oxidation. Since intramitochondrial oxidation of NADH is not rate limiting, the presence of labelled NADH in the cytosol may indicate that transfer of hydrogen through the mitochondrial membrane is a rate‐limiting step.As expected from the reversible reactions catalyzed by fumarate hydratase and malate dehydrogenase the deuterium excess at C‐2 and C‐3 of malate and fumarate were about the same, 20 atoms% Citrate had a deuterium excess of about 9 atoms%, assuming that only one hydrogen was labelled. Succinate contained very little deuterium. The labelling of citrate may thus be due either to incorporation of oxaloacetate labelled at C‐3 or to labelling of the same hydrogen in a reaction catalyzed by isocitrate dehydrogenase.The results indicate that both malate and isocitrate are potential sources of labelled NADPH during oxidation of [1‐2H2]ethanol, and that these two routes are not completely equilibrated under the experimental conditions.
Studies on Seed‐Oil Triglycerides Factors Controlling the Biosynthesis of Fatty Acids and Acyl Lipids in Subcellular Organelles of Maturing Crambe abyssinica Seeds
Authors: APPLEBY Robert S. , GURR Michael I. , NICHOLS Bryan W. 
Published: March 2005

The acetyl‐CoA carboxylase activity of all Crambe abyssinica subcellular fractions is very low as indicated by poor conversion of acetate and acetyl‐CoA and a requirement for malonyl‐CoA as the substrate for fatty acid synthesis.
Erucic acid (C22:1) biosynthesis is most active in the oil body fraction, quite active in the denser nuclear/plastid and mitochondrial fractions and absent from the microsomal and cytoplasmic fractions. Erucic acid is formed by direct elongation of oleic acid, not by synthesis from acetate de novo.
Desaturation of added stearyl‐CoA was not demonstrated.
Incorporation of 14C‐labelled acyl groups from fatty acyl‐CoA thioesters into phospholipids occurred in all subcellular fractions except the supernatant from centrifugation at 105000 ×g.
Triglyceride synthesis from 14C‐labelled stearyl‐CoA occurred in all subcellular fractions except the supernatant fraction from centrifugation at 105000 ×g.
Incubations of halved seeds with labelled precursors, followed by subcellular fractionation, supported these findings except that the intact seeds had functional acid‐CoA ligase and acetyl‐CoA carboxylase.
Incubation of halved seeds in the presence of trichloroacetic acid (2.5 mM and 12.5 mM) resulted in substantial inhibition of the biosynthesis of erucic acid but not of other fatty acids. Higher levels of trichloroacetic acid resulted in general inhibition of fatty acid and acyl lipid synthesis.
Modification of Phenylalanyl‐tRNA Synthetase from Escherichia coli by Histidine‐Specific Reagents
Authors: HENNECKE Hauke , BÖCK August 
Published: March 2005

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.
Study of Coenzyme Binding Site of Octopine Dehydrogenase Using Analogues of NAD+
Authors: OLOMUCKI Anna , THOMÉ‐BEAU Françoise , BIELLMANN Jean‐François , BRANLANT Guy 
Published: March 2005

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring.The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect.Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide–8‐bromoadenine dinucleotide, nicotinamide–8‐thioadenine dinucleotide and nicotinamide 1:N6‐ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes.Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding.The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary but not ternary complexes.The case of 1,4,5,6‐tetrahydronicotinamide–adenine dinucleotide which does not give ternary complexes like NADH is discussed.The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described.The protective effects of coenzyme analogues towards essential–SH group were also studied.
The Self‐Association of Chicken‐Erythrocyte Histones
Authors: DIGGLE John H. , McVITTIE John D. , PEACOCKE Arthur R. 
Published: March 2005

The self‐association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self‐association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non‐aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2a1 (H4) (highly aggregating).In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight‐average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self‐association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer‐dimer‐tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.
Interactions of Colipase with Bile Salt Micelles
Authors: SARI Hélène , ENTRESSANGLES Bernard , DESNUELLE Pierre 
Published: March 2005

The finding reported in the preceding paper that colipase is able to bind one sodium taurodeoxycholate micelle per molecule was confirmed by dialysis and spectrophotometry. Dialysis in the presence of labelled sodium taurodeoxycholate provided a direct qualitative proof of taurodeoxycholate binding to colipase. This binding was found to occur only above the critical micelle concentration. But, dialysis did not give any information about the composition of the associations, because equilibrium was not attained at the end of the assays.Addition of sodium taurodeoxycholate above the critical micelle concentration was also observed to induce a strong perturbation of the ultraviolet spectrum of one or several of the three tyrosines of colipase. The variation of the perturbation as a function of sodium taurodeoxycholate concentration was consistent with the binding of a single micelle to colipase. The dissociation constant calculated in “micelle molarity” was approximately 1 × 10‐4 M.The colipase‐bile salt micelle association can fix one molecule of lipase to form a ternary complex which represents an interesting model of a protein‐protein interaction mediated by an organized lipid structure. The ternary complex is probably also a model for lipase‐substrate interactions in the presence of an amphipath.
Purification and Characterization of a New Factor which Restores Protein Synthesis in a Conditionally Lethal Mutant of Escherichia coli
Authors: VAN DER MEER John P. , GANOZA M. Clelia 
Published: March 2005

The mutant Escherichia coli strain, N4316, has a temperature‐sensitive defect in a protein factor required for translation in vitro of bacteriophage f2 RNA. We have purified the normal counterpart of this factor from a wild‐type strain, using as an assay its ability to restore the activity of mutant extracts at non‐permissive temperature. Our final preparation is free of known initiation, propagation, and release factors, proving that the factor is a new component required for translation.The new factor has a molecular weight of 95000 with preliminary data suggesting a subunit structure. 70% of this protein is found in the soluble‐cell fraction, the rest being associated with 70‐S ribosomes. Kinetic analyses indicate that the factor acts early in translation.Expression of the defect is highly dependant on the Mg2+ concentration, no temperature‐sensitivity being apparent at 15 mM Mg2+. At lower Mg2+ concentrations, the defect is expressed only with natural mRNAs such as f2 RNA, and not with artifical polymers such as poly(U). This specificity suggests that the factor may function in events coded by special sequences in the natural messengers.
Evidence for an Essential Lysine in Glucose‐6‐Phosphate Dehydrogenase from Leuconostoc mesenteroides
Authors: MILHAUSEN Michael , LEVY H. Richard 
Published: March 2005

Pyridoxal 5′‐phosphate inhibits glucose‐6‐phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki= 0.04–0.06 mM.This inhibition is competitive with respect to glucose 6‐phosphate and non‐competitive with respect to NADP+ or NAD +. Interaction between enzyme and excess pyridoxal 5′‐phosphate follows pseudo‐first‐order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme.Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5′‐phosphate. Dissociation constants for NADP+ and glucose 6‐phosphate were determined from their effects on the kinetics of enzyme–inhibitor interaction.Reaction of the enzyme with pyridoxal 5′‐phosphate produces a typical Schiff‐base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine.The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N6‐pyridoxyllysine can be identified by paper chromatography.The enzyme is inhibited irreversibly by 1‐fluoro‐2,4‐dinitrobenzene, even in the presence of excess 2‐mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ and glucose 6‐phosphate protect against inhibition by 1‐fluoro‐2,4‐dinitrobenzene. The absorption spectrum of dinitrophenyl‐enzyme corresponds to that for dinitrophenylated amino groups.These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6‐phosphate.It is proposed that a group of “active lysine” proteins may exist (in analogy with the “active serine” enzymes), which share a common structural feature at their substrate‐binding site and to which pyridoxal 5′‐phosphate binds specifically.
Studies of Intracellular Thymidine Nucleotides
Authors: OHKAWA Tsutomu 
Published: March 2005

In a thymine‐deprived culture, the mutant cells (deficient in dTDP‐glucose pyrophosphorylase activity and named Ter‐15) lose viability at a faster rate, form longer filaments for the first 60 min and lose thymidine nucleotides and dTDP‐sugar pools at a faster rate for the first 15 min than those of the parent cells, but the dTDP‐sugar pool in the parent cells is maintained at high concentration for the first 90 min during thymine starvation.In the recovery of cell growth after re‐addition of thymine into the thymine‐deprived culture, parent cells recommence growth immediately, but the mutant cells (Ter‐15) show a lag‐phase for 45 min after which time their growth recommences.The rate of dTTP synthesis for the first 10 to 15 min after re‐addition of thymine to thymine‐deprived cultures of parent and mutant (Ter‐15) cells is three‐fold higher than that of a thymine nondeprived culture (control), but the rates of dTMP and dTDP‐sugar syntheses are the same as those of the control.The total DNA synthesis after re‐addition of thymine is equal to that of the control, and the period of thymine starvation other than the number of viable cells during thymine starvation plays an important role. After separation of the filament cells from normal‐sized cells by sucrose gradient centrifugation, the initial rate of DNA synthesis of filament cells is three‐fold faster than that of normalsized cells.These results show that the dependency of DNA synthesis upon dTTP concentration is maintained after re‐addition of thymine into thymine‐deprived culture.