bluuebunny/crossref_metadata_2025_split huggingface dataset:
Zn2+ and Co2+‐Alkaline Phosphatases of E. coli
Authors: Lazdunski C. , Lazdunski M. 
Published: March 2005

For the Zn2+‐phosphatase, the ratio k3/k2 (pH 8.4) of the dephosphorylation (k3) and phosphorylation (k2) rates is very similar, about 1.4–1.7, for substrates which are very different chemically such as p‐nitrophenyl phosphate, β‐glycerophosphate, 5′‐AMP and ATP. This means that k2 is nearly the same for all these substrates. On the other hand with o‐carboxyphenyl phosphate, phosphorylation is the determining step.
The Co2+‐enzyme has a much lower specific activity than the Zn2+‐phosphatase. The substitution of Zn2+ by Co2+ decreased the rate of phosphorylation by a factor of 6.4 for o‐carboxyphenyl phosphate and 16 for the above mentioned substrates; no such decrease is observed on k3. The phosphorylation is inhibited by nucleophilic agents which otherwise activate the Zn2+‐enzyme in the same conditions. The Co2+‐phosphatase offers a unique opportunity to compare the free energy of binding of the different substrates to form the Michaelis complex; those free energies are strikingly similar, they vary between – 6.1 and – 7.0 kcal. This is further evidence that specificity of the enzyme is not directed against the R part of RO PO3 even in such a peculiar case as ATP. Neither the Zn2+ nor the Co2+‐enzyme are able to catalyse the hydrolysis of a P‐N bond. Organic phosphates (P‐O bond) bind better to the phosphatase than p‐chloro‐anilido‐phosphonic acid (P‐N bond). The exchange of Zn2+ by Co2+ decrease only slightly the affinity for the active center of organic substrates or inhibitors; a factor of 1.78 for o‐carboxyphenyl phosphate and 2.65 for p‐chloroanilido‐phosphonic acid. The difference is more pronounced for inorganic phosphate; a factor of 23 (pH 9.16) and 27 (pH 8.5).
Two ionisable groups are important for the binding of the substrate to the active center. One must be in the basic form, it has a pK of 7.1 and 7.4 in the Zn2+ and Co2+‐phosphatase, respectively. The other must be acidic, the corresponding pK values are 8.6 and 8.9, respectively. With both metallo‐enzymes the phosphorylation step is pH‐independent between pH 6 and 9. The dephosphorylation step is dependent upon the ionization of a group which must be in the basic form and has a pK of 7.5 for the Zn2+‐phosphatase.
The enzyme remains dimeric in the course of the catalysis and it seems that there is only one active site phosphorylated per dimer.

k
2 and k3 are decreased in 2H2O indicating possibly rate‐determining proton transfers. The enthalpy and entropy of activation corresponding to both steps have been calculated.
Acetolactate Decarboxylase from Aerobacter aerogenes
Authors: Løken Jan P. , Størmer Fredrik C. 
Published: March 2005

Acetolactate decarboxylase has been highly purified from A. aerogenes.The purified enzyme does not need manganese for its activity as earlier described, and it is able to decarboxylate acetohydroxybutyrate, the second intermediate in the biosynthesis of isoleucine. The pH optimum in phosphate is 6.2–6.4.
Structure primaire de la caséine β bovine
Authors: Dumas Bruno Ribadeau , Grosclaude François , Mercier Jean‐Claude 
Published: March 2005

In order to determine the primary structure of the bovine β‐caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr).The tryptic hydrolyzate was separated by column chromatography on Dowex‐50. After additional purifications, 13 peptides numbered T1 to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino‐acid analyses were carried out on all these peptides. Peptide T1 contains 2 Arginyl residues, one of which is NH2‐terminal. This peptide represents the NH2‐terminal end of the β‐casein. Its composition is identical to that reported by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β‐casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2‐terminal. The COOH‐terminal peptide was identified with peptide T4, devoid of basic amino‐acids. The 14 tryptic peptides account for all the residues of the β‐casein A2 when compared with the already published amino‐acid analyses of the whole protein [2,3]. Several other peptides, originating from incomplete or non‐specific cleavages, were also obtained.Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β‐casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G‐50. The seventh peptide was obtained by chromatography on Dowex‐50 of the whole CNBr‐digest. After additional purifications, the amino‐acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2‐terminal end of β‐casein. The COOH‐terminal end of β‐casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr‐peptides account for all of the amino‐acid residues of β‐casein A2.β‐casein A2 has 208 amino‐acid residues. Its molecular weight is very close to 24000.
Regulierende Faktoren der Methylenblaukatalyse in Erythrocyten
Authors: Roigas H. , Zoellner E. , Jacobasch G. , Schultze M. , Rapoport S. 
Published: March 2005

In erythrocytes of man and rabbits methylene blue catalysis is limited by the following factors at the pH optimum 7.5; primarily by the cellular concentration of NADP+, next by that of glucose‐6‐P. Possible further limiting steps are glucose‐6‐phosphate dehydrogenase, phosphogluconate dehydrogenase and reduced‐NADP dehydrogenase.
Calculation of the intracellular NADP+ concentration with the two‐substrate equation for the glucose‐6‐phosphate dehydrogenase‐reaction does not yield reasonable results. The existence of inhibiting factors of the methylene blue catalysis is postulated. Their amount is presumably lower in intact cells as compared with hemolysates.
There is no pH dependence of the glucose‐6‐P concentration in presence of methylene blue. There exists a competition between the oxidative pentose‐phosphate‐pathway and the Embden‐Meyerhof pathway, in which the phosphofructokinase plays a key role.
The proportions of the two pathways at different pH values are calculated from data on oxygen and glucose consumption. At pH values below 7.6 glucose is utilized practically exclusively via the oxidative pentose phosphate pathway, whereas the Embden‐Meyerhof pathway is inhibited. At pH 8.2 the participation of the oxidative pentose‐phosphate pathway amounts to 40% of the total glucose utilization, while the Embden‐Meyerhof pathway is little if any reduced as compared to cells without methylene blue.
With increased degree of oxidation of the pyridine nucleotides there occur decreases in the levels of hexose‐ and triose‐phosphates. With these changes and the decreased flow to lactate an increased formation of 2,3‐diphosphoglycerate occurs.
Die Aminosäure‐Sequenz vom Cytochrom c des Karpfens, Cyprinus carpio
Authors: Gürtler L. , Horstmann H. J. 
Published: March 2005

Cytochrome c was isolated in pure form form hearts of the carp, Cyprinus carpio, by means of ion exchange chromatography and gel filtration. The protein had an iron content of 0.44% and showed an absorption spectrum characteristic of cytochrome c molecules. The ratio of the optical density at 550 nm of the reduced from to that at 280 nm of the oxidized form of the protein was 1.06 to 1.08.The protein was digested with trypsin or with a trypsin preparation which contained some chymotryptic activity. The resulting peptides were resolved on thin layer plates by electrophoresis and chromatography. The amino acid compositions of 40 peptides isolated from the fingerprints were analyzed. The sequences of 22 of these peptides representing the complete polypeptide chain of carp cytochrome c were determined.By comparing the peptides with the sequences of other vertebrate cytochromes c and by using the overlapping compositions of some peptides obtained by tryptic digestion or by splitting the protein with cyanogen bromide the complete sequence of 103 residues was deduced.Two types of cytochrome c have been found in our material differing at residue 4 of their sequences by glutamic acid (iso‐1‐cytochrome c) or aspartic acid (iso‐2‐cytochrome c). The origin of these two types is supposed to be a racial heterogeneity of carps used for human nutrition.Carp cytochrome c differs from cytochrome c of tuna in at least 8 residues, and from cytochrome c of the dogfish (Squalus sucklii) in at least 12 to 14 residues depending in this case on the location of a residue deleted in the cytochromes c of both bony fishes. Deviating from other vertebrates so far known the three species of fish have in common an asparagine at residue 22 and a serine at residue 54 of their cytochromes c.
Purification and Regulatory Properties of ATP‐Sensitive Phosphofructokinase from Yeast
Authors: Atzpodien W. , Bode H. 
Published: March 2005

Phosphofructokinase from baker's yeast has been purified 370‐fold. The purified enzyme is homogeneous as judged by analytical ultracentrifugation and disc electrophoresis. It exhibits the kinetic properties reported for phosphofructokinase from yeast and other sources. The purified enzyme is very sensitive to inhibition by ATP. The inhibition is pH‐dependent with a maximum around pH 7.5.The influence of different effectors on the enzymatic activity and on the inhibition by ATP are described. NH4+ ions cause a general stimulation of enzymatic activity, while the sensitivity to ATP is abolished. ADP inhibits the enzyme at non‐inhibitory ATP levels and stimulates the activity at inhibitory concentrations of ATP. Phosphoenolpyruvate is a negative effector. Inorganic phosphate inhibits the activity by competition with Fru‐6‐P and increases the inhibitory effect of ATP.
Molecular Weight and Quaternary Structure of Yeast l‐Lactate Dehydrogenase (Cytochrome b2)
Authors: Monteilhet C. , Risler J. L. 
Published: March 2005

An X‐ray diffraction study has been performed by the powder technique on two crystalline forms of yeast cytochrome b2. Type II (DNA‐free) cytochrome b2 crystallizes in the trigonal system, the parameters of the unit cell being a= 168.2 Å and c= 112.0 Å, whereas type I (DNA‐containing) cytochrome b2 crystallizes in the tetragonal system with a= 99.1 Å and c= 87.0 Å.Measurement of the crystal density and of the partial specific volume of the protein (found to be 0.754 cm3/g at 25°) enables a precise determination of the molecular weight of cytochrome b2 to be obtained. In this respect, the values 234600 ± 8000 and 235000 ± 10000 were found for type II and type I crystals respectively. These results, although in complete concordance, are quite different from the previously accepted value (approx. 180000 from sedimentation studies based on v= 0.71 cm3/g). Space‐groups and protein contents of the crystals show that this protein is composed of at least two, or even four, identical protomers.
Isolation and Characterization of Porcine Proelastase
Authors: Gertler Arieh , Birk Yehudith 
Published: March 2005

Porcine proelastase was purified from frozen pancreases by extraction at pH 4.5, precipitation with (NH4)2SO4, isolation of the pH 5.7 insoluble euglobulin fraction, selective (pH dependent) adsorption on elastin and chromatography on CM‐cellulose at pH 4.5. The active fraction was dialyzed and freeze dried. All the steps of purification except the last one were performed in presence of trypsin and chymotrypsin inhibitor AA from soybeans. All the process was done at 4°.
The proelastase thus obtained was found to be pure by acrylamide gel electrophoresis and ultracentrifugation. The extinction coefficient of the freeze‐dried material, E1cm1% at 280 nm, was 17.0. The pure preparation was free of α‐N‐benzoyl‐l‐arginine ethyl esterase and contained traces of pro‐N‐acetyl‐l‐tyrosine ethyl esterase. It was stable at pH 4–5 and inactivated by NaCl.
The molecular weight of the pure proelastase, calculated from the measured sedimentation and diffusion coefficients, was 25430 and according to the Yphantis method 25840.
Proelastase was activated by trypsin and by the trypsin‐like enzyme from pronase, the maximal activation being dependent upon the ratio of activating enzyme to proelastase. Elastase and peptidase A did not activate the proenzyme at all.
The similarity between proelastase and elastase was noted both in electrophoretic mobility in acrylamide gels and in amino acid composition but no NH2‐terminal could so far be detected in the proenzyme.
Cooperative Binding to Linear Biopolymers
Authors: Schwarz Gerhard , Balthasar Wolff 
Published: March 2005

The binding of acridine orange to poly(α‐l‐glutamic acid) at pH 7.5 has been investigated by means of absorbance measurements at 492 nm. The equilibrium as well as chemical relaxation behavior is interpreted in the light of a basic model theory of cooperative binding. Binding and cooperativity are found to be stronger than in the previously studied case of proflavine. In principle, the static properties are analogous in both cases. The only distinct contrast is given by the fact that the degree of cooperativity is considerably affected by ionic strength and polymer to dye ratio. This is attributed to the effect of unfavorable electrostatic interactions which tend to prevent long aggregates of bound dye.Temperature jump measurements resulted in relaxation curves which agreed very well with the predictions of the theory. Mean relaxation times of cooperative binding could be determined and evaluated in terms of an effective rate constant for recombination of free dye with bound aggregates of dye. The effect of varying the concentration of added KCl can be attributed to blocking of reaction sites by bound K+ ions. Extrapolation to zero KCl concentration yields a diffusion controlled rate constant for cooperative binding of monomer dye (kRo is approx. 1.5 × 109 M–1 sec–1). The life time of terminal elements of bound aggregates of dye turns out to be about 400 μsec.
Isolation and Characterization of the Cyanogen Bromide Fragments of a Mouse γG Immunoglobulin
Authors: Bourgois Alain , Fougereau Michel 
Published: March 2005

In order to isolate fragments suitable for amino acid sequence analysis, MOPC 173, a murine γG2a clonal immunoglobulin was cleaved with cyanogen bromide. On the basis of the methionine content of the molecule (2 residues for the light chains and 9 for the heavy chains), 13 fragments were expected to be released upon CNBr action. All 13 predicted fragments were isolated, either from the whole molecule or from its polypeptide chains and papain split Fab and Fc fragments. They were characterized by means of amino acid analysis, molecular weights as determined from gel filtration on Sephadex, NH2‐terminal analysis and COOH‐terminal determination. They accounted for the whole molecule.Chemical evidence allowed ordering of the light chain derived fragments. The heavy chain derived fragments were unequivocally assigned to the Fd or to the Fc portion of the chain. Three of them were positioned on the grounds of chemical evidence, the remainder beeing ordered by assuming homology with a human γG immunoglobulin of known sequence.
Regulation of Liver Pyruvate Kinase and the Phosphoenolpyruvate Crossroads
Authors: Llorente P. , Marco R. , Sols A. 
Published: March 2005

Certain kinetic properties of the pyruvate kinase activity in different tissues of the rat have been studied in fresh extracts with particular attention to their possible value for the regulation of gluconeogenesis and glycolysis.The pyruvate kinase activity in fresh extracts of the gluconeogenic tissues liver and kidney cortex has allosteric properties very marked in near physiological conditions, as follows: (a) co‐operativity in the kinetics with respect to the concentration of phosphoenolpyruvate; (b) strong allosteric inhibition by alanine and ATP, each of which raises the [S]0.5 value and increases the sigmodicity respect to the concentration of phosphoenolpyruvate; and (c) very strong activation by fructose diphosphate, which greatly lowers the [S]0.5 value and shifts the kinetics from markedly sigmoid to hyperbolic, and can fully counteract the inhibitory effects of alanine and ATP.Keeping the extracts of liver and kidney at low temperature (0–2°) leads to desensitization of the pyruvate kinase to the homotropic cooperativity of the phosphoenolpyruvate substrate and to the allosteric inhibition by alanine and ATP and the activation by fructose diphosphate. This cold desensitization is reversible. These facts and their time dependence make it possible to understand previous difficuties to obtain reproducible allosteric effects with the pyruvate kinase of rat liver.The pyruvate kinase of other tissues examined, including heart and adipose tissue, did not exhibit any of the allosteric properties above described for the enzyme of the gluconeogenic tissues.The μmolar range of fructose diphosphate concentrations required for activation of liver pyruvate kinase in the presence of the two physiological inhibitors and within the physiological range of concentrations of phosphoenolpyruvate fits well in order of magnitude with the calculated range of concentrations of free fructose diphosphate prevailing in liver.These results strongly support the hypothesis that an isoenzyme of pyruvate kinase present in liver and kidney has evolved with built‐in specific regulatory mechanisms in order to prevent the diversion of phosphoenolpyruvate from its way to glucose in glucoenogenic situations. An efficient regulation in the reversible switch over from glycolysis to gluconeogenesis seems to be feasible by the interplay of two feedback inhibitors, alanine and ATP, and a forward activator, fructose diphosphate.
Untersuchungen an Hämoprotein‐Modellen
Authors: Scheler W. , Mohr P. , Pommerening K. , Behlke J. 
Published: March 2005

In aqueous alkaline solution poly‐(N‐vinylimidazole) interacts with haemin to form two types of complexes. The molecular weight of the PVIm was about 12000 and the number of binding sites for haemin monomer varied between 8 to 17 per PVIm molecule in dependence on the pH. At pH < 11.0 a red haemin‐PVIm complex is formed showing absorption maxima at 532 and 411 mμ and at pH > 11.5 a green complex with maxima at 595, 578 and 391 mμ.
At pH < 9 the equilibrium between haemin and PVIm is nearly independent of pH but clearly dependent on the haemin concentration. This dependence as well as the asymmetry of the haemin‐PVIm association curves prove a dissociation of the haemin dimers into monomers in combining to PVIm.
The spectral type of the red complex and the characteristics of the complex formation at pH < 11 suggest a co‐ordinative interaction of one imidazole group of PVIm with the haemin iron. In the green compound above pH 11.5 there is no co‐ordinative linkage between PVIm imidazole residues and haemin iron or at most a very weak one.
Complex formation between N‐ethylimidazole or N‐methylimidazole with haemin is mainly due to enthalpy changes whilst the combination of PVIm with haemin is mainly related to entropy effects. The free enthalpies of the formation of the co‐ordinative red haemin‐PVIm complex and the non‐co‐ordinative green complex do not differ significantly. The stability of the haemin‐PVIm complexes can therefore be attributed mainly to non‐co‐ordinative interactions between both partners, probably to hydrophobic bonding.
The red haemin‐PVIm complex can bind one cyanide anion per haemin with high affinity. The spectrum of the resulting haemin‐PVIm‐cyanide complex shows absorption maxima at 542 and 419 mμ. Formation of the complex is independent of the pH, proving that the co‐ordinatively bound imidazole group is displaced from the iron by CN−. However, the haemin remains attached to the PVIm‐matrix.
The differences in complex formation of haemin with monomeric imidazole derivatives and with PVIm are essentially due to the existence of multiple non‐co‐ordinative interactions between PVIm and haemin. There are some analogies between this binding and the multiple non‐co‐ordinative bonds between haemin and apolar amino acid residues in hemoglobins.
Ionic Effects on the Ribosomal Quaternary Structure
Authors: Ghysen A. , Bollen A. , Herzog A. 
Published: March 2005

Escherichia coli ribosomes, in various ionic conditions, were examined by ultracentrifugation analysis and melting studies. It has been shown that:

The Tris ion acts as a monovalent cation and is indistinguishable from the potassium ion as far as its influence on the stability of the ribosome is concerned.
The ribosomal stability, as measured by the melting temperature (Tm), and the ribosomal conformation, as observed by ultracentrifugation analysis, are only dependent on R, the ratio of the mono to the divalent ions concentrations.
The evolution of the ribosome as R increases is characterized by three phases: in the first one, the ribosome is in its most compact form (70 S); in the second one, the ribosomal subunits undergo a progressive loosening; and in the third, they are subject to a stepwise unfolding.
The hinge between the first and the second phases corresponds to the ionic conditions required for the correct functioning of the ribosomes in protein synthesis in vitro, and also for successful ribosomal reconstitutions.
Étude du système multienzymatique hydrogène lyase chez Escherichia coli K 12 et ses mutants chlorate‐résistants
Authors: Azoulay Edgard , Marty Bernard 
Published: March 2005

The hydrogen lyase system of Escherichia coli K12 which decomposed formate to hydrogen and carbon dioxide involves two particulate enzymes, formate dehydrogenase and hydrogenase, and a soluble component.Formate dehydrogenase, as measured using phenazine methosulfate as electron acceptor, has been localized in particles bearing nitrate reductase activity. Formate dehydrogenase is synthesized throughout anaerobic growth and is not repressed by KNO3. This behavior is unlike that of the soluble fraction which does not appear until the end of exponential growth and which can only be considered as an electron carrier.Particulate preparations from cells of the E. coli K12 pleotropic mutants chl A− and chl B− do not have formic dehydrogenase activity, but if the supernatants of these same cells are added to particulate fractions of the wild type, the mixture produces H2 from formate. These results agree with our earlier observations suggesting that these pleiotropic mutations affect particle structure.
The Mode of Action of 5‐Aza‐2′‐Deoxycytidine in Escherichia coli
Authors: Doskočil J. , ŠOrm F. 
Published: March 2005

5‐Azadeoxycytidine is a good donor of deoxyribosyl group in reactions catalyzed by pyrimidine deoxyriboside phosphorylase, promoting the deoxyriboside‐dependent incorporation of thymine by wild‐type cells of E. coli about twice as effectively as either deoxycytidine or deoxyuridine. Prior deamination to 5‐azadeoxyuridine is necessary for the utilization of the deoxyribose moiety of 5‐azadeoxycytidine, since the deoxyribosyl‐donor activity is entirely lost in mutants deficient in cytidine deaminase. Upon infection of wild‐type cells with phage T4 the deamination of 5‐azacytidine and 5‐azadeoxycytidine is strongly depressed; a proportional loss of the deoxyribosyl‐donor ability of the latter is observed while the activity of deoxycytidine remains unaffected.In wild‐type cells of E. coli 5‐azadeoxycytidine inhibits protein synthesis and the replication of phage f2 as effectively as 5‐azacytidine, but has much less effect on the replication of phage T4. In deaminase‐less mutants 5‐azadeoxycytidine has practically no inhibitory effects.It is concluded that 5‐azadeoxycytidine enters the cells of E. coli via deamination, followed by phosphorolytic cleavage of the glycosidic bond. Direct utilization of the deoxynucleoside, conserving the glycosidic bond the 6‐aminogroup, does not occur to any appreciable extent. In accord with this finding the biological effects of 5‐azadeoxycytidine are similar to those of 5‐azauridine but different from the action of 5‐azacytidine.