bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Studies on the Metabolism of Steroids in the Foetus
Authors: Lisboa Belisário P. , Gustafsson Jan‐Åke
Published: March 2005

After incubation of testosterone with the 105000 ×g microsome fraction from human foetal liver 4‐androstene‐3α,17β‐diol was isolated. Incubation of testosterone with the 105000 ×g supernatant fraction resulted in the formation of 4‐androstene‐3α(and 3β),17β‐diol. When incubated with liver slices testosterone was metabolized into 4‐androstene‐3α,17β‐diol and 3α‐ and 3β‐hydroxy‐4‐androsten‐17‐one.
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.
Purification and Properties of Catechol 1,2‐Oxygenase from Trichosporon cutaneum
Authors: Varga Janos M. , Neujahr Halina Y.
Published: March 2005

A highly purified preparation of catechol 1,2‐oxygenase was obtained from a strain of Trichosporon cutaneum, isolated from soil as a predominant phenol utilizing yeast. The purified active enzyme was homogeneous upon polyacrylamide gel electrophoresis. The enzyme was gradually inactivated upon storage. The inactivation was followed by the appearance of a second, electrophoretically distinct band. The active enzyme is red, the inactive form is colourless. Its molecular weight is 109000 as determined by gel filtration; pH optimum 8.2. The Michaelis constants are 5.9 μM for catechol and 81 μM for oxygen. There is no cofactor requirement for activity. The enzyme is sensitive to iron chelating agents, reducing agents and quinones. The enzyme has a broad substrate specificity: besides catechol it is able to cleave hydroxyl‐ and methyl‐substituted catechols. Several catechol derivatives inhibit the enzyme activity towards catechol.
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.
Excretion of Steroid Hormones in Adults
Authors: Eriksson Håkan , Gustafsson Jan‐Åke , Sjövall Jan
Published: March 2005

The C19 and C21 steroids in faeces from pregnant women have been studied by gas chromatography‐mass spectrometry. Over 90% of the steroids identified were unconjugated and had the following general structures: androstane‐3,17‐diol, 3‐hydroxy‐17β‐pregnan‐20‐one, 3‐hydroxy‐17α‐pregnan‐20‐one, 3β‐hydroxy‐5‐pregnen‐20‐one, pregnane‐3,20‐diol, 3,16α‐dihydroxypregnan‐20‐one, 5β‐pregnane‐3α,17α,20α‐triol, pregnane‐3,16α,20‐triol, and 5α‐pregnane‐3,20α,21‐triol. The C21O2 steroid isomers in the monosulphate and “diconjugate” fractions had the 5α configuration whereas both 5α and 5β isomers were present in the free steroid fraction. The major part of the 5α‐steroids carried a 3β‐hydroxyl group whereas the predominant 5β‐steroids had a 3α‐hydroxyl group. The results suggest that steroids excreted in the bile are modified by hydrolase, oxidoreductase and dehydroxylase activities of the intestinal microorganisms. The amount of steroids in a pooled smaple of faeces from a nine‐month pregnant woman corresponded to a daily excretion of 33 mg. This value indicates that faecal excretion is important in the elimination of steroids in pregnancy.
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.
The Dissimilation of Higher Dicarboxylic Acids by Pseudomonas fluorescens
Authors: Hoet P. P. , Stanier R. Y.
Published: March 2005

This work provides presumptive evidnce that Pseudomonas fluorescens metabolizes the higher homologs (C6 to C10) of the saturated dicarboxylic acid series by β‐oxidation, mediated by a specific series of inducible enzymes. The initial step in the metabolism of four of these acids (pimelic, suberic, azelaic and sebacic acids) is the formation of the corresponding CoA‐ester. This step is mediated by a single, inducible thiokinase. A thiokinaseless mutant can utilize none of these four dicarboxylic acids. Although adipic acid can also apparently be activated to a slight extent by the thiokinase, the unimpaired ability of the thiokinaseless mutant to grow with adipate shows that there is a second mechanism for the activation of this member of the series. Adipic acid is also activated by an inducible adipate: succinyl‐CoA transferase, which cannot use the higher homologs of the dicarboxylic acid series as CoA acceptors (with the exception of pimelic acid, which is weakly activated). This transferase is probably responsible for adipic acid activation in vivo. Inducible dehydrogenases which can oxidise acyl‐CoA and β‐hydroxyacyl‐CoA derivatives of the higher dicarboxylic acids were also demonstrated, but their substrate specificities were not determined. The former enzyme can dehydrogenate both adipyl‐CoA and azelayl‐CoA; and the latter, β‐hydroxyadipyl‐CoA. A mutant unable to form the enzyme with β‐hydroxy‐adipyl‐CoA dehydrogenase activity cannot grow with any dicarboxylic acid of the C6 to C10 series.
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.
Interaction of Antibodies with Uridine Diphosphosphate Galactose 4‐Epimerase from Yeast
Authors: Olesen H. A. , Bertland A. U. , Kalckar H. M.
Published: March 2005

Antibodies from a rabbit immunized with uridinediphosphate galactose 4‐epimerase were shown to inhibit the specific activity of the enzyme and to precipitate [14C]UMP‐labelled epimerase.

It comprised about 0.33% of the immunoglobins and its Fab fragments had a binding constant of 0.2 × 109 M‐1 at 25° and 1.1 × 109 M‐1 at 4°. Despite the high energy of interaction the antibody did not liberate [14C]UMP from reduced [14C]UMP‐epimerase. It gave two precipitation lines both with the native and with the reduced epimerase in the immunoelectrophoresis. However, the same pattern was obtained with antibody from a UMP‐epimerase immunized rabbit that could not precipitate [14C]UMP‐epimerase or inhibit the enzymatic activity. It is not clear whether any of the lines represent the epimerase protein unit sensu strictu.
Steroids in Germfree and Conventional Rats
Authors: Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

Caecal contents from conventional rats has been shown to have steroid sulphatase and glucuronidase activities. Whereas the sulphatase activity was of microbial origin the glucuronidase activity was also present in caecal contents from germfree rats. In vitro incubations showed that sulphuric acid esters of the 3α‐, 3β‐, 17β‐ and 21‐hydroxyl groups were all hydrolyzed by the caecal bacteria. The ration between free and conjugated metabolites of [4‐14C]corticosterone administered to germfree and conventional rats was much higher in the conventional than in the germfree rats. This shows that microbial hydrolysis of steroid sulphates takes place also in vivo.
The Effect of Prednisolone Injected in vivo on RNA‐Polymerase Activities in Isolated Rat Thymus Nuclei
Authors: Drews Jürgen , Wagner Lutz
Published: March 2005

The capacity of isolated rat thymus nuclei to synthesize RNA in vitro was investigated under various experimental conditions. When incubated in the presence of MgCl2 alone thymus nuclei synthesized an RNA product which according to a nearest neighbour frequency analysis contains high proportions of dinucleotide sequences typical for ribosomal precursor RNA: GpC, CpG and GpG. When MgCl2 was replaced in the incubation mixture by MnCl2 and ammonium sulfate the nuclei formed a product of a more DNA‐like nucleotide composition. The time course of the incorporation of tritiated ribonucleosidetriphosphates into acid‐precipitable material was different in the two nuclear polymerase systems: while, in the presence of MgCl2, the incorporation of radioactive AMP or GMP reached a plateau after only 15 min of incubation, the reaction proceeded at a linear rate for at least 40 min in the presence of high concentrations of salt and MnCl2. The absolute amounts of radioactive RNA‐precursor material incorporated into RNA are considerably smaller in the Mg++‐activated system than in the presence of MnCl2 and ammonium sulfate.

The administration of prednisolone to adrenalectomized young rats was followed by a decrease in the ability of isolated thymus nuclei to synthesize RNA when MgCl2 was present. The relative frequencies of several dinucleotide pairs in the RNA synthesized by isolated thymus nuclei in the presence of MgCl2 changed significantly in response to steroid treatment. The adminstration of prednisolone, however, did not modify the quantity or the quality of the product formed by thymus nuclei in the Mn++/ammonium sulfate activated reaction. These results further indicate that glucocorticoids exert a preferential inhibitory effect on the formation of ribosomal precursor RNA in rat thymus cells.
The Influence of the 2′OH Group upon the Binding of Nucleotides by Basic Polyamino Acids
Authors: Arav Ruth , Wagner Karl G.
Published: March 2005

The position of the phosphate group on the ribose of nucleoside monophosphates markedly influences their binding behavior with basic polyamino acids, such as poly‐l‐lysine and poly‐l‐arginine. Equilibrium dialysis of adenine and guanine derivatives has revealed that the binding of 5′‐ and 3′‐ribonucleotides (group I, possessing a free 2′OH group) to basic polyamino acids is clearly different from that of 2′‐ribo and 5′‐deoxyribonucleotides (group II, the 2′OH group is blocked and absent, respect.). At low nucleotide to poly‐l‐lysine ratios, where the binding corresponds to the Langmuir isotherm, the binding constants of group I are smaller than those of group II. At higher nucleotide to polypeptide ratios the binding is cooperative and accompanied by a phase transition: guanine derivatives of group I show precipitation with poly‐l‐lysine at 0′, whereas with group II a gel‐like phase is separated; with poly‐l‐arginine only precipitation occurs. Analysis of the cooperative binding with guanine nucleotides and poly‐l‐arginine revealed that group I possesses considerably higher nearest neighbour interaction energies than group II. The stacking coefficients decrease in the order 3′‐GMP > 5′‐GMP > 5′‐dGMP > 2′‐GMP. The implications of these results are discussed.
A “Proportion Graph” Method for Measuring Binding Systems
Authors: Baulieu Etienne E. , Raynaud Jean‐Pierre
Published: March 2005

A new graphical methodology is proposed to visualize and estimate the binding at equilibrium of small molecules in protein mixtures, providing each binding system is independent and follows the law of mass action. In many cases, the plots of Lineweaver‐Burk or Eadie‐Scatchard are found to be unsuitable.

The method consists in plotting log [fraction bound] vs. log [total ligand]. The determination and statistical approximation of the binding parameters are performed and checked by means of a 360 IBM computer. Therefore instead of the linear method which may lead to arbitrary estimation, a non linear function covering the whole range of informations is used since nowadays computerized analysis is available.

Theoretical and experimental examples are given for biological samples containing one or two systems of high affinity (“specific”), in the presence or absence of a system of low affinity and of a relatively large number of binding sites (“non‐specific”). The experimental material consists of steroid hormones and plasma or target tissue proteins.
Excited States of Flavins Characterised by Absorption, Prompt and Delayed Emission Spectra
Authors: Gordon‐Walker Ann , Penzer Geoffrey R. , Radda George K.
Published: March 2005

The angles between the two principal long wavelength absorption oscillators of FMN and some other flavins have been measured by two methods. A novel technique involving dichroic bleaching gave a value (approx. 35°) consistent with that obtained from the polarisation of fluorescence extrapolated to infinite viscosity.

Flavins trapped in polyacrylamide films display E‐type delayed fluorescence and phosphorescence depending on the temperature. The variations in lifetimes and intensities of these two delayed emissions with temperature have been studied. The Arrhenius activation energies have been analysed in terms of a scheme that gives information about the interrelationships between different electronic states of flavins.
Purification from Calf Thymus of an Inhibitor of Deoxyribonuclease I
Authors: Lindberg M. Uno , Skoog Lambert
Published: March 2005

A protein inhibitor specific for pancreatic deoxyribonuclease (DNAase I) was purified 10‐ to 20‐fold from calf thymus. This purification resulted in a nearly homogenous protein demonstrating the relative abundance of the inhibitor among the soluble proteins of the crude extract.

Like the DNAase inhibitors from calf spleen described earlier the thymus inhibitor showed a high tendency to form high molecular weight aggregates of low specific activity. Conditions which decreased the rate of this self‐aggregation were found as were conditions for the dissociation of already formed inhibitor aggregates.

The molecular weight of the purified inhibitor was determined under both stabilizing and disaggregating conditions and values between 45000 and 50000 were obtained.

The interaction between the enzyme and the inhibitor was shown to give rise to a stable complex containing one molecule each of the involved protein species.
Isolation of Polypeptide Inhibitor of Phospholipase A from Cobra Venom
Authors: Braganca Beatriz M. , Sambray Y. M. , Sambray Roshan Y.
Published: March 2005

A polypeptide inhibitor of phospholipase A has been isolated from cobra venom (Naja naja). It is a basic protein with an isoelectric point above pH 8.6. The molecular weight is 5000. The inhibitor has been shown to form an inactive complex with the enzyme in the ratio of 1:1. The complex having a molecular weight of approximately 15000 was isolated using Sephadex G‐100. The complex disintegrated into the component units in starch gel electrophoresis suggesting the involvement of electrostatic bonds in its formation. It was observed that the basic proteins salmine, polylysine and cobra cytotoxin did not inhibit phospholipase A. The inhibitor did not combine with some of the basic proteins isolated from cobra venom. The inhibitor was specific for the phospholipase A and apparently interacts with groups which are involved in its catalytic activity.
Étude cinétique de changements conformationnels de la l‐glutamate déshydrogénase provoqués par le couple effecteur GTP + NADH
Authors: Jallon J. M. , di Franco A. , Iwatsubo M.
Published: March 2005

It is shown by rapid kinetics that when the coenzyme NADH and the effector GTP are added to the enzyme, l‐glutamate dehydrogenase, several changes occur following a multiphasic process. These changes are observed by a variation in the absorbance, and fluorescence intensity of the fixed nicotinamide ring of NADH, as well as by a variation in the fluorescence intensity of the fluorescent probe, ANS, which is fixed to the protein. By all three methods, two phases are generally observed, the slower phase (k2= 1.5 sec–1) being characterized by a change in the molar absorbancy coefficient of the nicotinamide ring (∼15% at 365 nm). The more rapid phase (k1= 60 sec–1) is characterized by a decrease in turbidity without any spectral modifications of the fixed NADH. The binding rates of the two ligands NADH and GTP, presented elsewhere, are too rapid to be taken into consideration as being responsible for the steps observed here.

The step (k1= 60 sec–1) which shows a decrease in turbidity is thus associated with a depolymerization of the polymer (composed of an indefinite number of units of molecular weight 310000). Although only partial, such a dissociation, already known to exist by simple dilution of the enzyme, proceeds with a slower rate (k= 12 sec–1) than that of the dissociation provoked by NADH and GTP.

A study was also carried out using the coenzyme NADPH instead of NADH. The amplitude of the phase identified with the depolymerization of the enzyme is similar to that observed for NADH. A striking difference is found in the fact that there is but a negligible modification of the nicotinamide ring of NADPH.

It should also be noted the order of incubation of these ligands affects only the rate of depolymerization; in particular, pre‐incubation of the enzyme with NADH, but not with NADPH, notably slows down the rate of depolymerization. The results obtained with NADH and NADPH give support to the hypothesis proposed by Frieden from kinetic studies, that NADH, not NADPH, can bind to two distinct sites, an active site and an effector or regulatory site. Correlation of our results with known kinetic data has enabled us to give a functional aspect to each site. The active site is associated with the depolymerization effect, while the regulatory site is the site which we consider to be responsible for the “protector” and “transconforming” effects.

A summary of the observed rates is presented in schematic form, in an effort to understand the mechanism of action of NADH and GTP. The process which culminates in the inhibition of the enzyme activity occurs before the two structural modifications which are measured here, since the inhibitory effect of GTP on the oxidation of the coenzyme appears in less than 2 msec. Preliminary results show that the activator ADP reverses both the depolymerization and the spectral modifications of NADH produced by NADH and GTP.
DNA Polymerase from Rat Liver Chromosomal Proteins
Authors: Howk Richard , Wang Tung Yue
Published: March 2005

Under standard assay conditions, the replicative DNA polymerase partially purified from rat liver non‐histone chromosomal proteins exhibited a marked preference for native DNA over heat‐denatured DNA as the template. However, work reported in the present paper demonstrates that this template preference could be reversed by changing the enzyme to template ratio in the assay system. This change in template preference was observed when either the concentration of DNA polymerase or of DNA was varied. Furthermore, it is shown that this alteration in template specificity is not the direct result of the action of an alkaline endonuclease present in the DNA polymerase preparation.
The Nucleotide Sequences of Tyrosine Transfer RNAs of Escherichia coli
Authors: Goodman H. M. , Abelson J. N. , Landy A. , Zadrazil S. , Smith J. D.
Published: March 2005

The tyrosine tRNAs specified by the E. coli amber suppressor gene suIII+ and the wild type non‐suppressing allele suIII− were selectively labelled with 32P in E. coli infected with transducing phage ψ80 carrying either of these genes. The determination of the nucleotide sequence of the suIII+ tyrosine tRNA is described. This tRNA has the anticodon CUA. The suIII− tRNA differs from suIII+ tRNA by a single nucleotide and has the anticodon where is a modified guanylic acid residue. E. coli su− cells contain two tyrosine tRNAs (I and II). The determination of the nucleotide sequence of these is described. Species I is identical to suIII− tRNA while species II differs by two nucleotides in the variable loop region.
Close Linkage between a Galactose Binding Protein and the β‐Methylgalactoside Permease in Escherichia coli
Authors: Boos Winfried , Sarvas Matti O.
Published: March 2005

It has been found that a gene necessary for the production of the galactose binding protein is closely linked, if not identical with the gene(s) determining the β‐methylgalactoside permease. This was determined by crossing a galactose binding protein negative, β‐methylgalactoside permease negative strain of E. coli K‐12 with wild type donor strains.

In addition, after mutagenation of a permease positive, binding protein positive strain of E. coli K‐12 with N‐methyl, N‐nitroso, N′‐nitroguanidine, some of the β‐methylgalactoside permease defective mutants showed concomitant changes in the galactose binding protein. Out of 28 independent permease defective mutants two had lost their ability to produce cross reacting material against antibodies specific for the galactose binding protein; four mutants showed temperature sensitive synthesis of cross reacting material; five mutants had immunochemically somewhat changed binding protein. The immunochemical assay showed no changes in the binding protein of the rest of the 17 mutants. However, the galactose binding activity of the crude extract of these mutants varied to a great extent (10–90%) from the activity‐found in the crude extract of the wild type.

These results suggest a close relationship between the galactose binding protein and the β‐methylgalactoside permease.
Circular Dichroism of DNA
Authors: Gratzer Walter B. , Hill L. Rowland , Owen Robert J.
Published: March 2005

The circular dichroism of sixteen DNA samples from fourteen species has been determined. Plots of molar ellipticity against base composition show considerable scatter, which is much too great to be accounted for in terms of reproducibility of materials and measurements. The data are most simply fitted with the assumption of linearity; extrapolation then leads to apparent circular dichroism curves for 0 and 100% (A + T). These are regarded as having probably no physical reality, but serve as standards for the calculation of circular dichroism curves for DNA of any given composition. For the materials used in the present work the discrepancy between the observed curves and those calculated from the formal standards is at worst not more than about 10% and much better than this over most of the range, particularly on the long‐wavelength side of the crossover. The use of a quadratic function, based on the assumption that the circular dichroism can be operationally expressed, like hypochromicity, in terms of three spectroscopic parameters, offers no significant advantage in describing the circular dichroism data.
Immunochemical Studies on Structural Proteins of the Red Cell Membrane
Authors: Furthmayr Heinz , Timpl Rupert
Published: March 2005

Up to one third of the membrane proteins from bovine, ovine and human red cells were solubilized similarily by water pH 9.5, aqueous pyridine, ATP‐mercaptoethanol solutions or urea pH 11. The proteins are heterogeneous but show some common features independent of the extraction method applied. The remaining insoluble proteins are rather different and contain most of the lipids and of the surface antigens.

In the extracts mainly a common membrane‐specific antigen of high anodic mobility was demonstrated which contains neither sialic acids nor lipids. This antigen is species‐specific and is not located on the membrane outside. It is not exposed to the surface after proteolytic treatment of intact red cells. The extracts are contaminated by traces of plasma proteins and surface antigens.

The membrane antigen can be correlated to a characteristic double band demonstrable in the stroma by disc electrophoresis in the presence of sodium dodecylsulfate. The molecular weights were estimated as 190 000 and 210 000, respectively. Evidence is provided that these components represent aggregates.
Preliminary Partial Purification of Hen Liver Microsomal Stearoyl‐CoA Desaturase
Authors: Gurr Michael I. , Robinson Malcolm P.
Published: March 2005

Hen liver microsomal stearoyl‐CoA desaturase has been prepared in “soluble” form, either by extraction of freeze‐dried microsomal fractions with high ionic strength phosphate buffer or by treatment with non‐ionic detergents. On a discontinuous density gradient, desaturase activity was recovered in the lightest fraction which is rich in lipids. On agarose gel, the desaturase activity was excluded in the void volume and therefore had a particle weight greater than 4 × 106. Removal of some of the lipids of the microsomal fraction led to inability to solubilize the desaturase, but resulted in enhancement of enzymic activity. Complete removal of lipids was not possible with acetone‐water mixtures and preparations from which the maximum amount of lipid had been removed contained one quarter to one half of the original enzymic activity. This residual activity could not be restored to the original value by addition of micellar dispersions of lipids.
Binding of Aldolase and Triosephosphate Dehydrogenase to F‐Actin and Modification of Catalytic Properties of Aldolase
Authors: Arnold Heidwolf , Pette Dirk
Published: March 2005

The binding of aldolase and triosephosphate dehydrogenase to F‐actin was studied in vitro under various conditions. Several monovalent and bivalent cations and anions, most of the glycolytic metabolites, and some nucleotides were examined with respect to their influence on the binding. Desorption of the bound enzymes as well as adsorption hindrance may be due to relatively specific factors (metabolites, ions) and relatively non‐specific factors (pH, ionic strength). In the case of aldolase it was found that fructose‐1,6‐diphosphate, dihydroxyacetone phosphate, ATP, ADP, and inorganic phosphate cause a marked adsorption hindrance within their physiological concentration ranges. In analogy with the effect of fructose‐1,6‐diphosphate on aldolase, the binding of triosephosphate dehydrogenase to F‐actin is also inhibited most strongly by its substrate glyceraldehydephosphate. However, adsorption hindrance of triosephosphate dehydrogenase is also caused by fructose‐1,6‐diphosphate, ATP, and inorganic phosphate, although higher concentrations of these compounds are needed in comparison with aldolase. Kinetic studies of F‐actin‐bound aldolase revealed that the properties of the enzyme are modified by the binding. Under the chosen experimental conditions, binding of aldolase to F‐actin doubles Vmax and increases Km for fructose‐1,6‐diphosphate by almost one order of magnitude. Km determinations were performed in the absence of ammonium sulfate. Sulfate and also phosphate anions are strong inhibitors of muscle aldolase. In both cases the inhibition type appears to be competitive. Determinations of Km for fructose‐1,6‐diphosphate of native muscle aldolase gave values as low as 0.4 μM.