bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational Properties and Catalytic Function of Aspartate Aminotransferase
Authors: Ivanov Valery I. , Bocharov Alexander L. , Volkenstein Michael V. , Karpeisky Marat Ya. , Mora Susanna , Okina Esther I. , Yudina Lyudmila V. 
Published: March 2005

Conformational stability of the aspartate aminotransferase has been studied. To this aim the isotherms of urea denaturation were obtained for the apoenzyme and the holoenzymes reconstituted with pyridoxal phosphate and its analogs. The state of the active site was shown to have an influence upon the general protein stability, the stability levels (the points of denaturation) were grouped aroud 5.1 M and 6.9 M urea depending on the analog structure.Proceeding from an idea of complementarity of free energy profiles in a cogruent model system and in the corresponding protein moiety, the conformational stability was determined of the aspartate aminotransferase · inhibitor complexes simulating the enzyme · substrate ones which are formed at the subsequent reaction steps. It was shown that a higher stablity of the protein corresponds to most of the high‐energy complexes of the congruent system. It is suggested that in the case of aspartate aminotransferase the equalizing of the energy profile along the reaction coordinate is realized by means of few (perhaps two) discrete energy states of the protein globule.
Studies on the Initiation of Plasmid DNA Replication
Authors: Goebel Werner 
Published: March 2005

The replication of the bacterial plasmids is dependent on two chromosome‐determined products (dnaA and dnaC), which are involved in the initiation of the chromosomal DNA synthesis of Escherichia coli. The inhibition of Col E1 DNA synthesis in a dnaA‐mutant (CRT 46) at the restrictive temperature, is eliminated by the addition of low doses of chloramphenicol (3 μg/ml). Under these conditions, a stimulated and relaxed synthesis of this plasmid is observed. The ability of chloramphenicol to overcome the inhibition of Col E1 DNA synthesis is dependent on the extent of the residual plasmid DNA synthesis at the time of addition of the antibiotic. Col E1 DNA synthesized at 43 °C in the presence of chloramphenicol, largely consists of supercoiled DNA circles containing RNA segments as revealed by their sensitivity to ribonuclease H of Escherichia coli and alkali. Addition of rifampicin to chloramphenicol‐treated cells causes first a slight inhibition of the relaxed Col E1 DNA synthesis at 43 °C and then a further stimulation.An immediate increase in plasmid DNA synthesis after the addition of rifampicin to chlor‐amphenicol‐treated cells at 43 °C is observed in the dnaA‐mutant carrying the antibiotic resistance factor R1. Another transmissible plasmid (Hly PM152) does not show this stimulated DNA synthesis. Stimulation of Col E1 and R1 DNA synthesis by rifampicin does not occur in chlor‐amphenicol‐treated cells of the dnaA‐mutant with a rifampicin‐resistant RNA polymerase. Chloramphenicol and/or rifampicin cannot eliminate the inhibition of plasmid replication at the restrictive temperature in a dnaC mutant (PC 2). In the presence of chloramphenicol, however, more complex Col E1 DNA molecules (dimeric and trimeric DNA forms) are synthesized at the restrictive temperature in the dnaC‐mutant.
The Effect of Adenosine Analogues on the ATP‐Pyrophosphate Exchange Reaction Catalysed by Methionyl‐tRNA Synthetase
Authors: Lawrence Françoise , Shire David J. , Waller Jean‐Pierre 
Published: March 2005

Following the finding that the ATP‐PPi exchange reaction and the aminoacylation of tRNA catalysed by methionyl‐tRNA synthetase from Escherichia coli are inhibited by adenosine, numerous analogues of adenosine have been examined to determine the nature of the specificity. The main features observed are as follows.

Modifications to positions 1, 2 and 6 of the purine ring of adenosine largely eliminate the inhibition of the ATP‐pyrophosphate exchange reaction.
The replacement of the nitrogen at position 7 with a carbon has virtually no effect.
Substitutions at positions 8 give analogues exhibiting a wide range of inhibition constants; 8‐aminoadenosine is a highly effective inhibitor, with a K1 300 times lower than that of adenosine.
The sugar part of the nucleoside is essential for binding, but can be considerably modified without too great a detrimental effect, particularly in the 5′‐position.
For the nucleosides which have a low inhibition constant the presence of a triphosphate group lowers the affinity, but for the analogues exhibiting a high inhibition constant a triphosphate group has no effect.
Steady‐State Kinetics of Rabbit‐Intestinal Sucrase
Authors: Semenza Giorgio , VON Balthazar Anna‐Karin 
Published: March 2005

Steady‐state analysis of product inhibition shows that the most probable kinetic mechanism of intestinal sucrase is ping‐pong bi‐bi (ordered uni‐bi for hydrolysis alone).
The site of activation by Na+ was tentatively localized at a level prior to the further transformation of the “glucose”· enzyme complex.
Tris(hydroxymethyl)aminomethane competes with the substrate for the glucose subsite.
In the appendix a simple kinetic text is described for detecting or ruling out a mutual competition between inhibitors of different or of identical kinetic type.
Adenosine and Inosine 5′‐triphosphates
Authors: Naumann Christoph F. , Prijs Bernhard , Sigel Helmut 
Published: March 2005

The chemical shift of the protons, H(2) and H(8), in the proton magnetic resonance spectra of ATP, ITP, and inosine was measured in aqueous solution in dependence on pH (2 to 12). Based on these data the sites of protonation or deprotonation are identified and the acidity constants determined. A comparison of the constants due to the nucleotides with the corresponding ones of the nucleosides evidences that the basicity of the nucleic base increases somewhat under the influence of the triphosphate chain. The possibility of an intramolecular hydrogen bound between N(7) and the proton of the γ‐phosphate group is discussed.From the line broadening effects in the presence of Cu2+ it is concluded that this metal ion interacts mainly with N(7) of ATP, but with the N(1), O(6)‐site of ITP. With inosine as ligand N(7) is the predominate binding site at pH <6, while at pH >7 N(1), O(6) is preferred. Experiments with ITP in the presence of [Cu2+] = [2,2′‐bipyridyl] (under conditions where Cu(Bipy)2+ is nearly completely formed) reveal the surprising fact that in the pH range 2 to 6 the line broadening of the signal due to H(8), and H(2) as well, increases significantly compared with a system containing only ITP and Cu2+. This observation is easily explained by the assumption of a chargetransfer interaction within the ternary complex, Cu(Bipy) (ITP)2‐, i.e. between the purine part of the nucleotide and 2,2′‐bipyridyl. This interaction could indeed be confirmed by spectrophotometric measurements in the ultraviolet (λmax= 313 nm). Similar results were obtained for the mixed‐ligand complex, Cu(Bipy) (ATP)2‐. The distance between the planes of the aromatic rings in these systems is estimated and a structure for these metal‐ion‐bridged charge‐transfer complexes is suggested. The biological implications of such ternary, metal‐ion‐bridged, charge‐transfer complexes are briefly indicated.
The Hydrolysis of p‐Nitrophenylacetate in Water
Authors: Goren H. Joseph , Fridkin Matitahu 
Published: March 2005

The hydrolysis of p‐nitrophenylacetate has been studied between pH 6 and 9 in 3.3%(v/v) dioxane‐0.1 M phosphate or Tris buffer. The results indicate that the hydrolysis is acid‐catalyzed, base‐catalyzed and has a pH‐independent rate component. The observed rate constants for the hydrolysis were determined spectrophotometrically from the initial rate of appearance of p‐nitrophenol (at 320 nm), p‐nitrophenoxide (at 400 nm) and the initial rate of disappearance of p‐nitrophenylacetate (at 270 nm). An equation has been derived permitting the calculation of first and second‐order rate constants from the initial slopes of product appearance or reactant disappearance. The semilog method of first‐order kinetic analyses could not be used since the hydrolysis of p‐nitrophenylacetate did not go to completion.The apparent incomplete reaction is attributed to the establishment of an equilibrium between p‐nitrophenol and p‐nitrophenyl esters. The latter probably includes p‐nitrophenyl phosphate. The rate constant for the synthesis of p‐nitrophenyl esters has been determined and it also has acid, base and pH‐independent catalytic components.
Subunit Interactions in Enzyme Catalysis
Authors: Ricard Jacques , Mouttet Christian , Nari Joannès 
Published: March 2005

The activation free energy of any chemical process, carried out by a polymeric enzyme, can be split up into various energetic components. The mathematical formulation of this splitting allows the writing of every phenomenological rate constant in terms of the corresponding intrinsic constant and interaction coefficients. Those interaction coefficients are dimensionless parameters simply linked to thermodynamic functions expressing the contributions of the various types of subunit interactions to the free energy of activation. With the usual formulation of Koshland only saturation functions can be written, whereas with the new thermodynamic formulation the expression (in terms of molecular parameters) of both the substrate‐binding isotherms and the steady‐state rate equations are made easy. The rate equations obtained are obviously dependent upon the “geometry” of the enzyme molecule and are, in fact, structural rate equations; that is, their formulation is changed on assuming different protomer conformations and interactions.These equations have been written for various models of subunit interactions (simple sequential, partially‐concerted, fully‐concerted, and exclusive allosteric) pertaining to dimeric and tetrameric enzymes.The saturation functions and the steady‐state rate equations, both expressed with the same structural formulation, have been compared for the above models. It appears, as a general rule, that it is impossible to reduce the structural rate equations to the corresponding saturation functions, even for systems occurring in a pseudo‐equilibrium state. The only exception to this general rule is the allosteric model with exclusive binding. It is thus impossible, with the exception of the above case, to describe velocity data with the concept of saturation function. This result is at variance with an assumption commonly made.The possible shapes of the plots relating the reciprocal of the rate to the reciprocal of substrate concentration, have been analytically established and simulated by computer, for the above models of subunit interactions in dimeric enzymes. It appears that with regard to the steady‐state velocity, subunit interaction can generate not only “positive” or “negative cooperativity”, but also “substrate inhibition”.
A Na+K+‐Activated Mg2+‐Dependent ATPase Released from Proteus L‐Form Membrane
Authors: Monteil Henri , Schoun Josiane , Guinard Marie 
Published: March 2005

The membrane‐associated, Na+K+‐activated, Mg2+‐dependent adenosine 5′‐triphosphatase activities of Proteus P18 L‐forms were further characterized. The enzyme was solubilized from membrane ghosts by repeated washings in a Mg2+‐free 33 mM Tris‐HCl buffer pH 7.5. Solubilized Mg2+‐dependent ATPase exhibits an initial increase in activity, followed by a fairly rapid inactivation at 0 °C. Proteus L‐form ATPase exhibits allotopic properties as seen in Micrococcus lysodeikticus, Streptococcus faecalis, Bacillus megaterium and Escherichia coli. The two monovalent cations (K+ and Na+) taken alone or together and the one divalent cation (Ca2+) were activators. The ATPase is highly specific for ATP as a substrate. ADP is a competitive inhibitor. N,N'‐Dicyclohexylcarbodiimide, a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation and of the membrane‐bound ATPase of Streptococcus faecalis, was found to inhibit both the membrane‐bound and the solubilized form of Proteus L‐form ATPase.
Conformational Changes and Proton Uptake in the Reversible Aggregation of Tobacco‐Mosaic‐Virus Protein
Authors: Vogel Dieter , Jaenicke Rainer 
Published: March 2005

The coat protein of tobacco mosaic virus is characterized by a complex circular dichroic spectrum in the range of aromatic absorption. Depending on the type of aggregate predominating under specific experimental conditions the various well‐resolved bands show positive or negative changes of their amplitude (Δɛλ≶ 0) which may be correlated quantitatively to corresponding structural transformations. A decrease of the amplitude is observed upon aggregating A‐protein to the double disc, while helix formation leads to a strong increase.Starting from the A‐protein at pH 8 (I= 0.1, 20°C), double discs may be formed either by decreasing pH or by increasing ionic strength; both methods of aggregation lead to identical changes in dichroic absorption at wavelength 265–290 nm, while at 295 nm only smaller changes occur.Following the time dependence of transconformation and aggregation by observing Δɛλ and by component analysis in the ultracentrifuge shows that both processes depend in a critical way on pH, ionic strength and the kind of ions applied. Under certain well‐defined conditions of the solvent the conformational changes precede the aggregation process.Correlating the temperature dependence of Δɛλ to that of acidimetric titrations shows a linear relationship between double‐disc formation, proton uptake, and change in dichroic absorption. The proton uptake amounts to 1 proton per monomer built into the double disc (I= 0.1).Including results from mutant studies of other authors, the wavelength of the different maxima of Δɛλ suggest tyrosine and/or tryptophan residues in the carboxylic end of the polypeptide chain to be involved in the conformational changes upon double‐disc formation.
Isolation and Study of the Composition of a Peptidoglycan Complex Excreted by the Biotin‐Requiring Mutant of Brevibacterium divaricatum NRRL‐2311 in the Presence of Penicillin
Authors: KegleviĆ Dina , Ladešič Branko , Hadžija Olga , Tomašič Jelka , Valinger Zdenka , Naumski Radmila , Pokorny Miroslav 
Published: March 2005

When cultures of biotin‐requiring mutant of Brevibacterium divaricatum NRRL‐2311 were treated in early logarithmic phase with penicillin (2–3 units/ml) in a glucose‐mineral medium under excess supply of biotin, large amounts of a high‐molecular‐weight material accumulated in the medium. The phenomenon could not be observed with cultures run in parallel from which penicillin was omitted. Chemical analyses of the excreted material isolated from the media of 1‐h and 24‐h penicillin‐treated cultures, showed that the main constituents were peptidoglycan components of noncross‐linked structure bearing both l and d‐alanine residues; evidence was also obtained for the occurrence of extractable lipid material, non‐amino sugars and organic phosphate.Under identical conditions, the excretion of peptidoglycan could be induced by ampicillin and cloxacillin, respectively, but not by bacitracin. Addition of penicillin to biotin‐requiring mutant of M. glutamicus yielded similar results, indicating that the phenomenon was not restricted to the Brev. divaricatum strain. This suggests that excretion of peptidoglycan material by the two biotin‐requiring mutants might be the result of two events, (a) a change in the osmotic barrier of the cell and (b) specific inhibition of cross‐link formation in peptidoglycan induced by penicillin.Several procedures were examined for the isolation and purification of the peptidoglycan complex excreted by penicillin‐treated Brev. mutant. Suitable labelling experiments with l‐[U‐14C]glutamic acid and analyses of lysozyme digests of the purified fractions, suggest that some of the components might contain murein fragments covalently linked to a polysaccharide portion.
A Study of the Density Pattern of ATPase and Respiratory Enzymes during Mitochondrial Biogenesis of Saccharomyces cerevisiae
Authors: Somlo Marie , Krupa Mireille 
Published: March 2005

Two different density patterns were found with ATPase‐carrying membrane populations from late stationary phase anaerobic and adapting cells of Saccharomyces cerevisiae (in the absence of growth). A membrane population at d= 1.23 g/ml carried the majority of the ATPase from anaerobic cells; it was still present in the case of adapting cells, but carried a decreasing proportion of ATPase, while increasing amounts of ATPase were now found in a lower‐density membrane population (d= 1.16 g/ml), absent from anaerobic cell extracts. Mitochondria from aerobically grown yeast also had a density of 1.16 g/ml in our experimental conditions.
The two ATPase‐carrying membrane populations from adapting cells could be distinguished by strikingly different ATPase/respiratory enzyme ratios: the membranes at d= 1.23 g/ml were enriched in respiratory enzymes, but relatively poor in ATPase, while the opposite was true for the membranes at d= 1.16 g/ml. In consequence, the ATPase/respiratory enzyme ratio was 2 to 3 times higher in the lighter than in the heavier peak.
Cycloheximide inhibited respiratory adaptation but the residual ATPase and respiratory enzymes nevertheless appeared in low‐density membranes (d= 1.16 g/ml). When chloramphenicol was present during anaerobic growth, not only respiratory adaptation was inhibited, but the residual ATPase and respiratory enzymes now remained in membranes of high density.
When exponential phase anaerobic cells were adapted, respiratory adaptation occurred, but ATPase and respiratory enzymes were distributed among high‐density membranes.
The influence of various experimental parameters and the possible physiological significance of the observed density patterns is discussed.
Oligomycin Sensitivity of ATPase Studied as a Function of Mitochondrial Biogenesis, using Mitochondrially Determined Oligomycin‐Resistant Mutants of Saccharomyces cerevisiae
Authors: Somlo Marie , Avner Philip R. , Cosson Jacky , Dujon Bernard , Krupa Mireille 
Published: March 2005

The quantitative comparison of oligomycin sensitivity of ATPase from two mitochondrially determined oligomycin‐resistant mutants (locus OI and OII) and the wild‐type strain (all three isonuclear), as a function of mitochondrial biogenesis, gave the following results:

The mutations at the loci OI and OII confer distinctly different oligomycin resistance on mitochondrial ATPase, OI being more resistant than OII, and both more resistant than the ATPase from the wild‐type strain.
In spite of these differences in oligomycin resistance, the binding of the ATPase to the mitochondrial membranes does not appear to be modified.
The phenotypic oligomycin resistance of mitochondrial ATPase, produced by growth of the wild type strain in the presence of chloramphenicol, is notably higher than the highest genotypic oligomycin resistance.
The oligomycin sensitivity or resistance of ATPase is strictly identical in anaerobic promitochondria and aerobic mitochondria, i.e. the difference between the oligomycin sensitivity of the ATPase from these strains is maintained in such extremely different physiological states.
In contrast, the ATPase isolated during respiratory adaptation of the wild‐type strain had distinctly higher oligomycin resistance than the promitochondria or the aerobic mitochondria. This “transient” resistance displayed by the wild‐type ATPase during adaptation (in the absence of growth) was very similar to the genotypic resistance of the ATPase from the mutant at locus OI.
Comparisons of the Tryptophan Synthase Inactivating Enzymes with Proteinases from Yeast
Authors: Saheki Takeyori , Holzer Helmut 
Published: March 2005

The partially purified tryptophan synthase inactivating enzymes I and II were compared with the earlier described proteinases A, B and C by studying their catalytic properties, their sensitivity against a serine proteinase inhibitor and their temperature stability.

Tryptophan synthase inactivase activities of inactivase I and II coincided with hemoglobin hydrolyzing and azocoll hydrolyzing activities, respectively, on hydroxyapatite column chromatography.
Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were insensitive to the treatment with phenylmethylsulfonyl fluoride, whereas inactivase and azocoll hydrolyzing activities of the inactivase II fraction were almost completely inhibited by phenylmethylsulfonyl fluoride.
The N‐acetyltyrosine ethyl ester (Ac‐Tyr‐OEt) hydrolyzing activity of an inactivase I fraction from an hydroxyapatite column could be separated from the inactivase activity by DEAE‐Sephadex column chromatography. Because of its esterolytic properties, the separated fraction corresponds to proteinase C.
Inactivase and hemoglobin hydrolyzing activities of the inactivase I fraction were stable at pH 6.0 and 37°C, but unstable at pH 8.0 and 37°C, while inactivase and azocoll hydrolyzing activities of the inactivase II fraction were unstable even at pH 6.0 and 37°C.

From these studies it is concluded that inactivase I is identical to proteinase A, inactivase II is identical or very similar to proteinase B and that proteinase C had no tryptophan synthase inactivase activity.
Immunochemical and Enzymic Properties of the Electric Eel Acetylcholinesterase‐Antiacetylcholinesterase System
Authors: Gurari Dalia , Silman Israel , Fuchs Sara 
Published: March 2005

Antibodies to acetylcholinesterase from the electric organ of the electric eel were elicited in rabbits by immunization with the different molecular forms of the enzyme, namely, the globular 11‐S and the “native” 14‐S + 18‐S acetylcholinesterase.The antigenic specificity of the two acetylcholinesterase preparations was analysed by the reaction of antisera to each of them with both the homologous and heterologous enzyme antigens. The immunological techniques employed included precipitation, immunoelectrophoresis and complement fixation. No differences in immunodominant antigenic determinants were observed between the two forms of the enzyme. The difference in the pattern of reaction between the 11‐S and 14‐S + 18‐S preparations with either antibody, in both the precipitation reaction and complement fixation, is ascribed to the higher aggregative state of the “native” 14‐S + 18‐S acetylcholinesterase preparation.The effect of antibodies to acetylcholinesterase on the enzymatic activity of the 11‐S and 14‐S + 18‐S preparations towards the substrates acetylcholine, acetylthiocholine and indophenyl acetate was studied. The enzymic activity towards acetylcholine and acetylthiocholine is markedly inhibited by specific antibodies whereas the activity towards indophenyl acetate is hardly affected under similar conditions. The monovalent fragments, Fab, of the antibodies have no inhibitory effect on acetylcholinesterase activity. From kinetic studies it appears that the antigenic determinants involved in enzyme‐antibody interactions are not located in the active site, and that the inhibition caused by antibodies under certain experimental conditions results from microenvironmental effects on enzyme activity.