bluuebunny/crossref_metadata_2025 huggingface dataset:
The Binding of Indole Derivatives by Borohydride‐Reduced β‐Cyclopiazonate Oxidocyclase
Authors: Steenkamp Daniël J. , Schabort C.
Published: March 2005

Initial velocity studies on β‐cyclopiazonate oxidocyclase in the presence of the competitive inhibitor, 3‐acetyl‐5‐scatyl‐tetramic acid, indicates that this enzyme catalyzes the oxidocyclization of β‐cyclopiazonic acid by a Ping‐Pong‐Bi‐Bi mechanism.

It was possible to reduce the flavin coenzyme to the 3,4‐dihydroflavin by consecutive cycles of reduction with BH4‐ and reoxidation by atmospheric oxygen.

The BH4‐‐reduced enzyme shows a similar affinity for indole derivatives as does the native flavoquinone enzyme, but is inactive. β‐Cyclopiazonic acid binds to the enzyme in a simple equilibrium, and the reduction of the 3,4‐dihydroflavin to the 1,3,4,5‐tetrahydroflavin or a conversion to 1,5‐dihydroflavin does not proceed.

Partial reactivation of the enzyme by the photocatalyzed oxidation of the 3,4‐dihydroflavin to the flavoquinone form was observed
Studies of Sugar Transport in Acholeplasma laidlawii
Authors: Tarshis Mark A. , Migoushina Valentina L. , Panchenko Leonid F. , Fedotov Nikolay S. , Bourd Heinrich I.
Published: March 2005

Different mechanisms of carbohydrate transport into Acholeplasma laidlawii cells have been studied. The following properties have been found.

The glucose, maltose and fructose uptake is dependent on the time of incubation, the substrate concentration in the external medium, as well as on the pH and temperature, thus pointing to the enzymatic nature of carriers.

The carbohydrate uptake is inhibited by a number of energy inhibitors and uncouplers, thus pointing to the active nature of this proces.

The carbohydrate uptake is highly sensitive to SH‐inhibitors, characterising carriers as proteins.

The noncompetitive inhibition suggests that glucose and maltose are transported by single carrier involving two active centres.
A New Immobilized NAD+ Analogue, Its Application in Affinity Chromatography and as a Functioning Coenzyme
Authors: Lindberg Margareta , Larsson Per‐Olof , Mosbach Klaus
Published: March 2005

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6‐carboxymethyl‐NAD+. Condensation of this analogue with 1,6‐diaminohexane gave the analogue NAD+‐ N6‐[N‐(6‐aminohexyl)‐acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50–100%.

NAD+‐N6‐[N‐(6‐aminohexyl)‐acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three‐enzyme system (malate dehydrogenease‐citrate synthase‐lactate dehydrogenase). The immobilized NAD+ analogue proved to be and effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively.
Further Characterization of Glycogen from Type‐IV Glycogen‐Storage Disease
Authors: Mercier Christiane , Whelan William J.
Published: March 2005

Glycogen from a male child suffering from type IV glycogenosis was debranched with bacterial isoamylase and fractionated on Sephadex G‐50. The unit chains, set free by the isoamylase, emerged as a single peak, similar to that for normal human liver glycogen, but of higher average chain length. The pattern of the unit chains was distinctly different from that shown by amylopectin, which displays two populations of unit chains. It is thereby reaffirmed that, contrary to previous belief, type IV glycogen is not amylopectin‐like in structure, and the name “amylopectinosis” for this disease is inappropriate.
The Rôle of Cytochrome P‐450 in Cholesterol Biosynthesis
Authors: Gibbons Geoffrey F. , Mitropoulos Konstantinos A.
Published: March 2005

The rates of cholesterol biosynthesis were studied in rat liver microsomal preparations incubated with [14C]lanosterol in the presence of gas mixtures of either carbon monoxide + oxygen (90:10, v/v) or nitrogen + oxygen (90:10, v/v). The presence of CO in the gas mixture resulted in a considerable decrease in the amount of cholesterol synthesized, and there was also a reduction in the radioactivity of the 4α‐methyl sterol fraction. The maximum rate of cholesterol biosynthesis in the presence of either N2+ O2 or CO + O2 occurred when the concentration of the substrate [14C]lanosterol was greater than 25 μM. The rate of cholesterol biosynthesis was constant for at least 40 min irrespective of the nature of the gas phase. During each time period studied, however, the amount of cholesterol formed was greater in the presence of N2+ O2. Removal of either ATP or NADPH from the incubation medium resulted in a significant decrease in the rate of microsomal cholesterol biosynthesis. Pre‐treatment of rats with phenobarbital resulted in a 35–40% increase in the rate of microsomal cholesterol biosynthesis from lanosterol. Incubations carried out under CO + O2 in which the flasks were illuminated with high intensity light of around 450 nm wavelength completely reversed the inhibitory effect of CO on cholesterol biosynthesis from lanosterol.
Thymine Utilization in Escherichia coli K12 on the Role of Deoxyribose 1‐Phosphate and Thymidine Phosphorylase
Authors: Jensen Kaj Frank , Leer Johan Chr. , Nygaard Per
Published: March 2005

Exogenously supplied thymine is only poorly utilized by wild‐type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine.

Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1‐phosphate and/or the level of thymidine phosphorylase.

Thymine incorporation in wild‐type cells could be observed if the cells possessed an increased deoxyribose 1‐phosphate pool.

The kinetics of thymine uptake in wild‐type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration.

The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1‐phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that thymidine is synthesized from deoxyribose 1‐phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside.
A Study of the Interaction of Calcium Ions with Bovine αs1‐Casein Using Fluorescence Spectroscopy
Authors: Dalgleish Douglas G.
Published: March 2005

The interaction of αs1‐casein with calcium ions at pH 7.0 has been studied, and it is shown that there is a stepwise increase in the fluorescence of the tryptophan residues of the protein as increasing amounts of Ca2+ are added, leading to eventual precipitation of the calcium‐caseinate complex.

The first of these steps appears to result from a conformational change induced by Ca2+, without which the protein cannot precipitate. Further steps arise from interaction between protein molecules to produce particles of about 120 nm in diameter, then final aggregation of these particles to give a precipitate. All of these changes result in changes in the fluorescence emission spectra of one or both tryptophan residues in the casein, although most of the change in fluorescence behaviour in the first step of the interaction probably arises from tryptophan‐164 in the protein:
ω‐Oxidation of Stearic Acid in the Normal, Starved and Diabetic Rat Liver
Authors: Björkhem Ingemar
Published: March 2005

ω‐Oxidation of stearate, catalyzed by 20000 ×g supernatant fluid of rat‐liver homogenate in the presence of an NADPH‐generating system, was stimulated three‐ to seven‐fold by starvation or diabetes. The soluble fraction of liver homogenate was responsible for the stimulation. Thus, addition of 100000 ×g supernatant fluid of liver homogenate from starved rats stimulated microsomal ω1‐oxidation of stearate two‐ to three‐fold whereas addition of 100000 ×g supernatant fluid of liver homogenate from untreated rats had no effect or an inhibitory effect. Evidence was obtained to indicate that the stimulatory effect of the 100000 ×g supernatant fluid from liver of starved rats was due to the presence in this fraction of liver alcohol dehydrogenase and NAD. The role of alcohol dehydrogenase might be to protect the ω1‐hydroxylase from product inhibition by oxidation of the 18‐hydroxystearic acid formed. The effect of 100000 ×g supernatant fluid from liver of untreated rats was shown to be due to an inhibition of the ω1‐hydroxylase reaction and not of the alcohol dehydrogenase reaction. The inhibitory effect of the 100000 ×g supernatant fluid was retained after boiling for 5 min but was partially destroyed after preincubation at 37°C or after repeated freezing and thawing. The inhibition was shown to be at least not mainly due to interference with formation of stearoyl‐CoA from stearic acid. Stearoyl‐CoA was ω1‐hydroxylated by the microsomal fraction or by the 20000 ×g supernatant fluid at a rate about half that of free stearic acid.

The possibility is discussed that the low extent of ω1‐oxidation of long‐chain fatty acids observed under normal conditions might be partially due to the presence of the inhibitor(s) in the soluble part of the cell and that disappearance of this inhibition under conditions of starvation and diabetes might be of physiological importance.
Mutants of Bacillus subtilis Blocked in Acetoin Reductase
Authors: López Juan , Thoms Brigitte , Fortnagel Peter
Published: March 2005

The reduction of dichlorophenolindophenol in the presence of 2,3‐butanediol can be used as a histochemical indicator for the acetoin reductase catalyzed reaction in viable Bacillus subtilis cells. Using this reaction it was possible to identify mutants blocked in this enzyme. The mutation which causes a lack in acetoin reductase simultaneously causes a loss of diacetylmethylcarbinol reductase activity and a reduction of diacetyl reductase activity. It was shown that the mutants lack one enzyme protein which carries all three activities. The residual diacetyl reductase activity originated from a different enzyme which could be separated from the protein with the multiple activities. The mutation did not affect acetoin degradation, however it prevented butanediol utilization.
Effect of Adenosine on Methionyl‐tRNA Synthetase
Authors: Lawrence Françoise
Published: March 2005

The methionyl‐tRNA synthetase activities were shown to be inhibited by adenosine. In the ATP⇋PPi exchange, and in the hydroxamate reactions, the inhibition was competitive with respect to ATP and uncompetitive with respect to L‐methionine. In the methionine transfer reaction, inhibition patterns are more complicated, however the inhibition tends to be competitive with respect to ATP at high methionine concentration.

The inhibition by adenosine of the ATP⇋PPi exchange reaction has been observed for several aminoacyl‐tRNA synthetases (seryl‐, cysteyl‐, isoleucyl‐, valyl‐, phenylalanyl‐, methionyl‐ and trypsin‐modified methionyl‐tRNA synthetases). However under standard conditions the degree of inhibition varies from one enzyme to the other.
Studies on the Association Behaviour of Human‐Erythrocyte Phosphofructokinase
Authors: Zimmermann Gerolf , Wenzel Klaus Wolfgang , Gauer Joachim , Hofmann Eberhard
Published: March 2005

By applying analytical gel chromatography technique the association behaviour of human erythrocyte phosphofructokinase has been studied. The molecular weight distribution of the polymerized forms of this enzyme covers a range of 380000 to about six millions. The degree of polymerization has been found to depend on the protein concentration, on the presence of various substrates and effectors and on the pH‐value. ATP favours the aggregation of the enzyme with increasing pH, whereas in presence of fructose 6‐phosphate or fructose 1,6‐bisphosphate respectively the degree of association appears to be independent of the pH value between pH 6 and 9. In the microgram range the specific activity of the enzyme decreases with an increase of the enzyme concentration.
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.
Preparation of the Enzymatically Active Subfragment of Myosin by Proteolysis of Myofibrils
Authors: Biró Endre N. A. , Coelho Rose , Ehrilich Elisabeth , Guillain Florent , Dvonc Christiane
Published: March 2005

Myofibrils, when submitted to proteolysis, yield several fragments, some of which remain bound to actin. There are separated by pyrophosphate treatment and isolated by preparative centrifugation. When trypsin treatment is performed in the presence of 1 mM CaCl2, two types of products are obtained, which can be separated by chromatography on DEAE‐cellulose. However, when proteolysis is performed with 10 mM EDTA in the medium, only one enzymatically active fragment is isolated. It is characterized by its sedimentation coefficient, α‐helix content, amino‐acid composition, actin‐binding properties in the absence of ATP and actin‐activation of its enzymatic activity. It is thus shown that the EDTA type fragment is similar to heavy‐mero‐myosin subfragment 1 obtained by direct treatment of myosin or heavy meromyosin with papain or trypsin.
Two Types of Vesicles from the Erythrocyte‐Ghost Membrane Differing in Surface Charge
Authors: Heidrich Hans‐G. , Leutner Gerhard
Published: March 2005

Vesicles of erythrocyte ghosts were produced by treatment with Primaquine or hexokinase. The resulting vesicles could be separated from each other and characterized by preparative free‐flow electrophoresis. One population consisted of outside‐out vesicles carrying the same sialic acid distribution as the original ghosts. The other represented homogeneous inside‐out particles with a different electrophoretic mobility carrying most of their sialic acid at their inner side. The two kinds of vesicles could not be separated by density gradient centrifugation.
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.
Changes of the Conformation of Rabbit IgG Antibody Caused by the Specific Binding of a Hapten
Authors: Pilz Ingrid , Kratky Otto , Karush Fred
Published: March 2005

The conformation of rabbit antibody specific for the ρ‐azophenyl‐β‐lactoside group was studied by small‐angle X‐ray scattering and the effect of interaction with hapten evaluated. The binding of hapten to the antibody caused a change in the conformation of the protein. The experimental findings indicate that the radius of gyration and the volume of the antibody become smaller by 2 to 3% as a result of interaction with hapten when 50% of the combining sites are occupied. Since the typical shape of the scattering curves remains the same it is inferred that the change of conformation consists mainly of a volume contraction whereas the overall shape, which is best described by T‐shaped models, is not essentially modified.
Prostaglandin F2α Receptor in Ovine corpora lutea
Authors: Powell William S. , Hammarström Sven , Samuelsson Bengt
Published: March 2005

Evidence is presented for the existence of a prostaglandin‐F2α receptor in ovine corpora lutea. Prostaglandin F2α was specifically bound to a particulate fraction of this tissue with a dissociation constant of 0.1μM. Prostaglandins F1α, E1 and E2 had considerably less affinity for the receptor whereas prostaglandins A2 and B2 had little or no affinity. From a comparison of the binding constants of various prostaglandins and related compounds it could be concluded that the carboxyl group, the three hydroxyl groups and the 5,6‐cis double bond (but not the 13,14‐transdouble bond) of prostaglandin F2α were all important for binding to the receptor.
The Sequential Inactivation of Glycogen Phosphorylase and Activation of Glycogen Synthetase in Liver after the Administration of Glucose to Mice and Rats
Authors: Stalmans Willy , De Wulf Henri , Hue Louis , Hers Henri‐Géry
Published: March 2005

The use of anesthetized animals and of quick‐freezing techniques has allowed to obtain a reliable estimation of the level of phosphorylase a in the liver. A striking inverse relationship was observed between the levels of phosphorylase a and of synthetase a in the liver of fed mice; this observation is in agreement with the hypothesis that the inactivation of phosphorylase is a prerequisite to the activation of glycogen synthetase. Accordingly, the administration of glucose to fed rats caused an extensive inactivation of liver phosphorylase which was usually terminated within two minutes; glycogen synthetase became activated only when and if the level of phosphorylase a had been taken down below a threshold value equal to approximately 10% of the total phosphorylase. This threshold is of the same magnitude in mice. Glucose also caused a slight decrease in the activity of liver phosphorylase when the animals had been previously treated with glucagon. These observations are adequately explained by the previously described stimulation of the phosphorylase phosphatase reaction by glucose and inhibition of synthetase phosphatase by phosphorylase a. In some experiments, insulin caused a partial inactivation of phosphorylase in the liver of rats but more often was without effect, whereas a subsequent load of glucose caused the usual precipitous response, demonstrating that this effect of glucose was not mediated by insulin.
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.
Protein‐Detergent Interactions
Authors: Yonath Jacob , Blauer Gideon
Published: March 2005

Interactions of sperm whale ferrimyoglobin with the cationic detergent laurylpyridinium chloride in aqueous medium were studied by measurement of spectral changes in the Soret band of the prosthetic group. Spectrophotometric titrations were carried out in 0.02 M sodium phosphate buffer pH 7.1 at 25 °C in the concentration ranges of 1 mM—1 μM myoglobin and up to 10 mM laurylpyridinium chloride and also at various NaCl concentrations, up to 0.5 M. A thermodynamic analysis was carried out assuming in all‐or‐none interaction between the hemoprotein and the detergent. The apparent stoichiometric coefficients for the association of the detergent with the protein and the average standard free energy changes of the interactions were evaluated. The thermodynamic data obtained correspond to values characteristic for hydrophobic interactions. The dependence of the spectral transition curves on total myoglobin concentration found in the present system enabled an evaluation of equilibria involving dissociation of the prosthetic group from the ferrimyoglobin by the action of the cationic detergent. Direct experimental evidence obtained for this process by chromatography has been presented previously. This leads to interpretation of the spectral effects observed as a result of association between ferriheme and laurylpyridinium chloride molecules.
The Allosteric Activation of Mammalian α‐Amylase by Chloride
Authors: Levitzki Alexander , Steer Michael L.
Published: March 2005

α‐Amylase from hog pancreas is shown to possess one binding site for Cl− per molecule of enzyme with a dissociation constant of 3 × 10−4 at 25 °C. The chloride anion functions as an activating effector increasing kcat 30‐fold towards either starch or p‐nitrophenylmaltoside. No change in Km for either substrate is seen. The other monovalent anions Br−, I−, NO2−, NO3−, ClO4−, SCN−, N3− and CNO− can substitute for chloride but their effect on kcat decreases as their anionic radius increases. Chloride binding induces a subtle conformational change in the enzyme reflected by the suppression of the exchange of 26 protons and a 240‐fold increase in the amylase‐Ca2+ binding constant from 8.3 × 108 to 2 × 1011 M−1. The conformational change is minor since it is not detected by circular dichroism, protein fluorescence or by specific probes attached to the enzyme. The ability of small structural changes to induce large catalytic acceleration is discussed.
Murein Hydrolases in the Envelope of Escherichia coli
Authors: Hartmann Rainer , Bock‐Hennig S. Barbara , Schwarz Uli
Published: March 2005

Properties of murein hydrolases, assumed to play an essential role in bacterial morphogenesis, are described. Evidence is presented that in the intact cell these enzymes are prevented from uncontrolled reaction. It is shown that the “barrier” between the enzymes and their substrate sacculus, a shape‐maintaining macromolecule within the cell envelope, can be artificially broken. Assay systems to measure murein hydrolase activity in the intact cell, in isolated cell envelopes and after solubilization are described. A combination of Triton X‐100 and sodium chloride is shown to liberate membrane‐bound hydrolases effectively.
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.
Affinity Chromatography on Immobilised Adenosine 5′‐monophosphate
Authors: Lowe Christopher R. , Harvey Michael J. , Dean Peter D. G.
Published: March 2005

The effect of column geometry and dynamics on the interaction of glycerokinase and lactate dehydrogenase with N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose has been studied. The strength of the enzyme‐ligand interaction and the capacity of the adsorbent were shown to be complex functions related to column geometry, ligand concentration and total amount of ligand. The bed geometry had a significant effect on the strength of the interaction at low ligand concentrations and a particularly pronounced effect on interacting systems of relatively low affinity. Enzyme retardation was dependent upon ligand concentration and was independent of the concentration of both the applied enzyme sample and inert protein. The enzyme‐ligand interaction, under column conditions, was shown to be a time‐dependent process and evidence is presented to show that both the resolution and strength of interaction can be enhanced by increasing the enzyme‐ligand equilibration time. Under batchwise conditions the apparent capacity of the adsorbent was dependent on both the enzyme concentration and the equilibration time. No difference was detected in the capacity of N6‐(6‐aminohexyl)‐5′‐AMP‐Sepharose for glycerokinase and lactate dehydrogenase under batchwise conditions.
A Sequence of 54 Nucleotides from the A‐Protein Cistron of Coliphage‐R17 RNA
Authors: Rensing Ulrich F. E. , Coulson Alan , Schoenmakers John G. G.
Published: March 2005

Three fragments from the A protein cistron of coliphage R17 ribonucleic acid have been found, purified and analysed from a partial T1 ribonuclease digest. A sequence of 54 nucleotides common to all three of them has been established. Two possible amino‐acid sequences were derived from the nucleic acid sequence by the genetic code, one of which must be part of the A protein.
Enzymes Involved in Wall Peptide Crosslinking in Escherichia coli K12, Strain 44
Authors: Nguyen‐Distèche Martine , Ghujysen Jean‐Marie , Coyette Jacques , Pollock Jerry J. , Reynolds Peter , Perkins Harold R. , Salton Milton R. J.
Published: March 2005

By using the glutamate‐amidated tetrapeptide l‐alanyl‐d‐isoglutaminyl‐(l)‐meso‐diamino‐pimelyl‐(l)‐d‐alanine as a probe, there appears to exist in the membranes of Escherichia coli K12 strain 44 a dd‐carboxypoptidase‐transpeptidase system which does not recognize this peptide and a dd‐carboxypoptidase‐transpeptidase system which recognizes it. The dd‐carboxypeptidase‐endopeptidase system is essentially hydrolytic. It catalyzes the hydrolysis of UDP‐N‐acetyl‐muramyl‐pentapeptide into UDP‐N‐acetylmuramyl‐tetrapeptide and the hydrolysis of the wall peptidoglycan peptide dimer into monomers. These activities are not inhibited by the glutamate‐amidated tetrapeptide. The system may consist either of two enzyme proteins having predominantly carboxypeptidase activity and endopeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. The dd‐carboxypeptidase‐transpeptidase system (a) catalyzes concomitant hydrolysis (carboxypeptidase activity) and transfer (natural model transpeptidase activity) reactions with the pentapeptide l‐alanyl‐γ‐d‐glutamyl‐(l)‐meso‐diaminopimelyl‐(l)‐d‐alanyl‐d‐alanine. The transfer reaction leads to the synthesis of a dimer that is identical to the one which occurs in the E. coli wall peptidoglycan; (b) utilizes the glutamate‐amidated tetrapeptide as an acceptor. Simultaneous exposure of the pentapeptide and the glutamate‐amidated tetrapeptide to the enzyme system leads to the formation of an hybrid monoamidated peptide dimer and causes a decreased hydrolysis of the pentapeptide; (c) by virtue of its own carboxypeptidase activity, it appears to exert some endopeptidase activity. Both carboxypeptidase and endopeptidase activities of this system are inhibited by the glutamate‐amidated tetrapeptide, but this represents only a small fraction of the total hydrolytic activity of the membrane Brij‐36T extract. (d) The system catalyzes an unnatural model transpeptidation reaction in which glycine replaces d‐alanine at the C‐terminal position of the nucleotide UDP‐N‐acetylmuramyl‐pentapeptide. This system may also consist either of two enzyme proteins having predominantly natural model transpeptidase activity and unnatural model transpeptidase activity, respectively, or of one enzyme protein of which the functioning would depend upon the environmental conditions. Whatever the exact situation, the E. colidd‐carboxypeptidase‐transpeptidase system is in many respects, similar to the dd‐carboxy‐peptidase‐transpeptidase single polypeptide enzymes isolated from Streptomyces strains R39 and R61.
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.
Interaction of Phosphofructokinase with the Fluorescent Probe 2‐(N‐Methylanilino)naphthalene‐6‐sulphonate
Authors: Hoffr Hans Werner , Radda George K.
Published: March 2005

The interaction of phosphofructokinase from rabbit skeletal muscle with 2‐(N‐methylanilino)‐naphthalene‐6‐sulphonate was studied. The fluorescence maximum of the probe was shifted to 430 nm and was enhanced by the enzyme. The intensity of emitted light was reduced by the presence of either one of the substrates, fructose 6‐phosphate or ATP. The reduction of fluorescence by fructose 6‐phosphate was accompanied by a decrease in the number of binding sites for the probe on the protein. The number of binding sites for the probe was also dependent on the concentration of the enzyme. Decrease of enzyme concentration resulted in relatively higher fluorescence and in a rise of the number of binding sites. Phosphofructokinase is inactivated after dilution as well in the absence as in the presence of the probe as a consequence of dissociation into inactive subunits. In the presence of the probe, the loss in activity followed the same kinetics as the increase in fluorescence. The rise in the number of probe binding sites, therefore, is most probably due to the dissociation of the enzyme. The effect of substrates on fluorescence intensity was used to follow interactions of the enzyme with its substrates. At low substrate concentrations the fluorescence changes were dependent on the concentration of the enzyme. At higher enzyme levels, higher ATP but lower fructose 6‐phosphate concentrations were required to obtain half‐maximal effects, thus suggesting that the binding of the substrates is dependent on the association state of the enzyme.
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.
Enzyme and Enzyme Fragments Purifiation by Specific Immunoadsorbents
Authors: Guiso Nicole , Truffa‐Bachi Paolo
Published: March 2005

Chromatography of crude extracts of Escherichia coli K‐12 on an immunoadsorbent column prepared with antibodies directed against aspartokinase I‐homoserine dehydrogenase I and elution with 6 M urea results in the one‐step isolation of the denatured protein. The same immunoadsorbent has been used for the purification of a protein extracted from a nonsense mutant, carrying only the aspartokinase activity, characterized by a shortened polypeptide chain. The tryptic maps of the two proteins are identical with those of the same proteins obtained by conventional methods. The application to protein sequencing is discussed.
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.