bluuebunny/crossref_metadata_2025 huggingface dataset:
The Specific Binding of Zinc(II) to Alkaline Phosphatase of Escherichia coli
Authors: Csopak H.
Published: March 2005

The binding of Zn2+ to Escherichia coli alkaline phosphatase has been studied by pH titrations and equilibrium dialysis using 1,10‐phenanthroline as competing chelating agent.

Measurements of the strength of binding under different experimental conditions have been performed:

At constant pH with different concentrations of chelating agent.

At different pH with constant concentration of chelating agent.

In the presence of denaturing agent at pH 8.0.

It was found on the basis of equilibrium dialysis experiments that the binding of the two zinc atoms to alkaline phosphatase may be described as coordination to the two equivalent and independent sites.

The high values of the binding constants, and the fact that denaturation destroys the specific binding of zinc to alkaline phosphatase indicates chelate formation with the protein, unless some unknown prosthetic groups are involved.
The Effect of Prednisolone Injected in vivo on RNA Synthesis in Rat Thymus Cells
Authors: Drews J.
Published: March 2005

Three hours after the injection of prednisolone into rats a strong reduction in the incorporation of [3H]uridine and [3H]cytidine into the acid precipitable material of thymus cells suspended in a tissue culture medium is observed. The inhibition of the incorporation of RNA precursors appears to be a specific effect of glucocorticoids. It is not caused by a dilution of the precursor pool or by an acceleration of RNA breakdown. These conclusions can be drawn from experiments in which the precursor pool was expanded by a factor of 100 and from kinetic experiments with actinomycin D respectively. A series of DNA‐RNA hybridization experiments were conducted to answer the question whether prednisolone causes the loss of certain species of hybridizable RNA. No such effects could be found. However, inhibition experiments with low doses of actinomycin, hybridization experiments performed at low RNA/DNA ratios, and pulse‐label experiments with [32P]orthophosphate suggest that prednisolone, besides causing a general inhibition of RNA synthesis, exerts a more specific preferential reduction in the formation of ribosomal RNA.
Histones from Sperm of the Sea Urchin Arbacia lixula
Authors: Palau J. , Ruiz‐Carrillo A. , Subirana J. A.
Published: March 2005

Whole histone and five fractions were prepared from the sperm of the sea urchin Arbacia lixula. They were characterized by starch gel electrophoresis and amino acid analyses. The fractions are classified as follows: ϕ1 (very lysine‐rich), ϕ2b (lysine‐rich), ϕ2a2 (intermediate type), and ϕ2a1 and ϕ3 (lysine‐poor). They have many similarities with calf thymus histone fractions, F1, F2b, F2a2, F2a1 and F3, in this order. Nevertheless, differences exist, the most important being a higher arginine content in several fractions, and the high proportion of the very lysine‐rich fraction found in the sperm of A. lixula.
Klassifizierung der Pullulanase als Exoenzym mit Hilfe der Gaschromatographie1
Authors: Wallenfels K. , Rached I. R. , Hucho F.
Published: March 2005

Pullulanase from Aerobacter aerogenes is a glucosidase specifically splitting α‐1,6‐glycosidic bonds. A polymeric substrate for the pullulanase is the linear α‐glucan pullulan from Pullularia pullulans.

This α‐glucan consists of maltotriose units linked together by α‐1,6‐glycosidic bonds. This means that the polysaccharide is degraded by pullulanase to yield exclusively maltotriose. There are two possibilities of enzyme action:

a) either the hydrolysis starts from one end of the linear chain directly setting free maltotriose;

b) or random splitting occurs to give larger units of varying molecular weight which will finally be degraded to maltotriose.

The decision between these two alternatives could be made by gas liquid chromatography. The quantity of liberated maltotriose rises linearly with the reaction time, which proves that hydrolysis occurs from the end of the substrate chain. This method using gas liquid chromatography as an analytical tool may also be applied to monitor the action of other hydrolytic enzymes, as it could be shown for α‐ and β‐amylase.
Malate Dehydrogenases of Ox Kidney
Authors: Dupourque D. , Kun E.
Published: March 2005

Kinetics of malate dehydrogenases of ox kidney were studied by means of three experimental models: bisubstrate kinetics, product inhibition and use of a powerful competitive inhibitor, β‐monofluoro‐oxalacetate. Application of Dalziel's treatment of kinetic parameters and Cleland's interpretations of product inhibition as well as criteria of competitive inhibition by fluoro‐oxal‐acetate yielded results which exclude reaction mechanisms other than compulsory ordered addition. Both isozymes exhibit identical idealized kinetics, provided anomalies of the mitochondrial enzyme (due to high degree of sensitivity to ionic environment) are simplified by experimental design. Anomalies of kinetics of the mitochondrial enzyme, caused by changes in ionic environment are probably consequences of conformational alterations.
The Substrate Specificity of Pigeon Liver NAD Kinase
Authors: Apps D. K.
Published: March 2005

The specificity of pigeon liver NAD kinase for both substrates and for divalent cations has been investigated. 2. The enzyme is activated by Mg++, Co++, Mn++, and Zn++ only: the ATP‐metal complexes have different Km, values but the maximum velocities of the reactions are the same. 3. All nucleoside triphosphates investigated will act as substrates, although the Km values and maximum velocities differ. 4. The only NAD analogue found to act as a substrate was 3‐acetyl pyridine‐adenine dinucleotide (APAD). 5. The enzyme is inhibited by free Mg and free ATP. 6. The independence of substrate binding sites confirms that a random‐addition, rapid equilibrium mechanism applies to NAD kinase.
Iodation des protéines par voie enzymatique
Authors: Nunez J. , Pommiter J.
Published: March 2005

In the preceding publication we have shown that horse radish peroxidase forms an enzymehalogen complex with iodide; first the halogen is oxidised by the enzyme forming the complex; secondly the oxidised halogen is transfered directly from the complex to acceptor proteins. Therefore we have assumed that an interaction may occur between the enzyme‐halogen complex and the tyrosine residues of the protein acceptor.

In the present work we will show that an enzyme‐protein complex can be formed; when horse radish peroxidase is incubated in the presence of both thyroglobulin and an H2O2 generating system, thyroglobulin isolated after the reaction is linked to peroxidase molecules. This complex is not formed when the H2O2 generating system is omitted. This result suggests that the tyrosine groups of thyroglobulin are oxidised by the peroxidase. Free tyrosine is indeed oxidised by the enzyme through a free‐radical mechanism; the fact that 50% of the radioactivity, present in the 3,5 positions is freed as tritiated water indicates that [3,5‐3H2]tyrosine forms a dimer during peroxidase oxidation. If free tyrosine is oxidised by such a mechanism we might assume that the residues of the same amino acid linked to a protein may be used as a substrate by the peroxidase. Such an assumption is supported by the fact that proteins are competitive inhibitors of the gaïacol peroxidative oxidation. If the tyrosine residues of the protein are oxidised as free radicals we might assume that the oxidised form of iodine linked to the enzyme is I. Therefore the mechanism of enzymatic iodination of proteins should proceed by an addition of free radicals.

We can conclude from kinetic studies that the presence of an increasing amount of protein, up to a certain limit, inhibits the iodination reaction. Iodide inhibits only when it is present in excess, where I2 is formed. From these results we might propose the following mechanism for the iodination reaction: the enzyme contains two sites almost identical; each of them should be able to form the complex with one of the two substrates, sequentially; when iodide is linked first, the velocity of the reaction attains maximum; when the protein is linked prior to the first site, iodide linkage to the second site is slowed and the velocity of the reaction reduced.
Affinité pour la streptomycine des ribosomes d'Escherichia coli
Authors: Petitpas‐Dewandre A. , Barbason H. , Verly W. G.
Published: March 2005

Ribosomes were isolated from four Escherichia coli K12 mutants, obtained by the action of ethyl methanesulfonate–with high (R) and low (r) resistance to streptomycin, dependent on high (D) and low (d) concentrations of streptomycin–and from the original sensitive strain (S). The ribosomal preparations were analyzed by sucrose‐gradient centrifugation, analytical ultra‐centrifugation, electron microscopy, determination of RNA to protein ratios, ability to synthesize polyphenylalanine in the presence of poly U, and the effect of streptomycin on this synthesis.

The amounts of the antibiotic taken up by the ribosomes of the sensitive strain and of the mutants were determined for different concentrations of free [14C]streptomycin and the results plotted according to Scatchard's equation. The graphs show that each ribosome, in the mutants as well as in the sensitive strain, contains only one receptor site for one streptomycin molecule; as measured by the equilibrium constant of the reversible adsorption, the affinities of the binding site for the antibiotic are: S = 109 × 103; r = 13 × 103; R = 3.8 × 103; d = 42 × 103; D = 10 × 103.

The observation that the ribosomes from the selected dependent mutants as well as those from the simply resistant ones have lower affinities for streptomycin than the ribosomes from the sensitive strain, could mean that the ribosomes from the resistant and the selected dependent mutants do not belong to different categories, and that E. coli dependence on streptomycin might have an extraribosomal origin.
Purification and Properties of DNA‐Dependent RNA‐Polymerase from Bacillus stearothermophilus
Authors: Remold‐O'Donnell E. , Zillig W.
Published: March 2005

The purification of DNA‐dependent RNA polymerase from Bacillus stearothermophilus is described. The enzymes from three different strains of this organism are compared with the Escherichia coli polymerase. The physical and enzymological properties appear very similar to those of the E. coli enzyme. The heat stability is considerably higher.
Der Testosteronstoffwechsel in der isoliert perfundierten Meerschweinchenleber
Authors: Demisch K. , Staib W.
Published: March 2005

Testosterone metabolism of the guinea pig was investigated on the isolated perfused liver. Eight experiments were carried out using four male and four female animals, respectively.

We could isolate and quantitate seven metabolites which showed identical chromatographic behaviour with corresponding reference substances such as free steroids, steroid‐acetates and steroid‐trimethylsilyl‐ethers on thinlayer plates and on two different stationary phases during gas‐liquid‐chromatography.

The identified metabolites constituted 73% of the radioactive material extractable by organic solvents after perfusion.

The major part of the remaining 27% of radioactive material was found on the starting point of the first chromatogram. These metabolites are likely to be hydroxylated derivatives of dihydro‐ and tetrahydro‐testosterone because of their polar behaviour. Recently such metabolites could be identified.

It was shown that the liver of the guinea pig hydroxylates testosterone and androstenedione mainly in position 6β‐ and 7α. The reductive metabolism of testosterone was very low. Only 3α‐hydroxy‐5β‐androstane‐17‐one and 3α,17β‐dihydroxy‐5β‐androstane were isolated in very small amounts. No metabolites giving evidence for 5α‐reductase activity were found.

Sex differences in the metabolism of testosterone could not be demonstrated.
Biosynthesis and Metabolism of Hydroxyphenylacetic Acids in Higher Plants
Authors: Kindl H.
Published: March 2005

2‐Hydroxyphenylacetic acid, a natural phenolic product found in the genus Astilbe, derives from the shikimic acid pathway via phenylpyruvic acid. The existence of two routes for the biosynthesis of 2‐hydroxyphenylacetic acid could be demonstrated:

a) A direct transformation of phenylpyruvic acid into 2‐hydroxyphenylacetic acid involving a migration of the side chain. More than 95% of the tritium activity of 2‐hydroxyphenylacetic acid was localized in position 5 when l‐[4‐3H]phenylalanine was fed. This complex oxidation is analogous to the known conversion of 4‐hydroxyphenylpyruvic acid to homogentisic acid.

b) A hydroxylation of [4‐3H]phenylacetic acid to [4‐3H]2‐hydroxyphenylacetic acid was observed in vivo, and was also found to take place in vitro utilizing the system peroxidaseendiol‐O2.

2,3‐Dihydroxyphenylacetic acid and 2‐hydroxy‐3‐methoxyphenylacetic acid could be established as natural products occurring in higher plants. Their chemical synthesis is described. By feeding experiments the following metabolic pathway is suggested: 2‐hydroxyphenylacetic acid → 2,3‐dihydroxyphenylacetic acid → 2‐hydroxy‐3‐methoxyphenylacetic acid. 3,4‐Dihydroxyphenylacetic acid and 3‐methoxy‐4‐hydroxyphenylacetic acid have been detected in extracts from various species of Astilbe and identified by paper chromatography. These two acids are metabolic products of 4‐hydroxyphenylacetic acid.

Experiments with [U‐14C]shikimic acid and dl‐[α‐14C]phenylalanine seem to indicate that the regulation of the biosynthesis of 2‐hydroxyphenylacetic acid and 4‐hydroxyphenylacetic acid takes place on the level of prephenic acid.

Preliminary results were obtained consistent with the hypothesis that in Astilbe chinensis the 2,3‐dihydroxyphenylacetic acid, 3,4‐dihydroxyphenylacetic acid and 2,5‐dihydroxyphenylacetic acid are further metabolized and can be degraded by ring cleavage.
Studies on the Morphopoiesis of the Head of Phage T‐Even
Authors: Kühl P. W. , Hofschneider P. H.
Published: March 2005

Assembly of T4 polyheads and T4 phage has been investigated under special metabolic conditions of the host cell. The purpose of the investigation was to determine whether the assembly of polyheads or capsids depends on protein and DNA synthesis and whether it is energy requiring. The influences of chloramphenicol, 5‐fluoro‐2′‐deoxyuridine and potassium cyanide on the maturation process were studied. The main results of experiments performed with T4D.20 (amN50) and T4D.20(amN50)‐e are as follows:

The maturation of polyheads and capsids is independent of concurrent protein synthesis; if the protein synthesis is arrested 10 min after infection or later, the existing protein precursor pools can be depleted by 90%.

With phage DNA synthesis reduced to about 10%, the capsid assembly is diminished, but not that of polyheads. Capsids may, polyheads may not need DNA as a morphopoietic factor.

The assembly of polyheads, but not that of capsids is partially inhibited by cyanide which lowers the intracellular ATP level to 10–20%.
Biochemical Studies on Lipopolysaccharides of Salmonella R Mutants
Authors: Gmeiner J. , Lüderitz O. , Westphal O.
Published: March 2005

The endotoxically active cell wall glycolipid of a highly deficient heptose‐less R mutant of Salmonella minnesota, consisting of 2‐keto‐3‐deoxyoctonic acid (KDO) and lipid A, was subjected to hydrazinolysis. Besides fatty acid hydrazides, a water‐soluble main fraction was obtained, containing KDO, glucosamine, and ester phosphate and free phosphorylethanolamine. The application of two procedures of partial hydrolysis, followed by paper electrophoretic separation, led to seven oligosaccharides.

Their structures were evaluated with the aid of the following analytical methods: further acid degradation, borohydride reduction, ninhydrin degradation, the Morgan‐Elson reaction, treatment with phosphomonoesterase and N‐acetylglucosaminidases. The results show that the backbone of lipid A contains phosphorylated glucosaminyl→glucosamine disaccacharide units, probably linked β‐1,6, to which a trisaccharide composed of three KDO residues is linked ketosidically to form a pentasaccharide which appears to be a common structure in many Enterobacteriaceae.
Zur Bildung von Chinolinalkaloiden in Pflanzen
Authors: Luckner M. , Winter K. , Reisch J.
Published: March 2005

Cyclopenase, an enzyme from the mycelium of Penicillium viridicatum Westling and Penicillium cyclopium Westling catalyses the transformation of the benzodiazepine derivatives (–)‐cyclopenine and (–)‐cyclopenol to the quinoline derivatives viridicatine and viridicatol, respectively. This rearrangement, which is connected with the elimination of molar amounts of CO2 and methylamine takes place in an nonenzymatic process too, if (–)‐cyclopenine and (–)‐cyclopenol are treated with acids or Lewis acids. In anhydrous medium instead of CO2 and methylamine, methylisocyanate is obtained.

14CO2 is formed from (–)‐[5‐14C]cyclopenine either by the enzyme or by the acid‐catalysed transformation, the viridicatine being inactive (Table). In experiments with 2H2O or H218O as solvents neither the cyclopenase nor the acid‐catalysed rearrangement proceeded with a significant incorporation of 2H or 18O (Fig. 1).

These experimental results indicate that:

a) enzymatic and nonenzymatic transformations of the investigated benzodiazepines to the corresponding quinoline derivatives proceed by similar mechanisms,

b) closure of the hetero‐ring of the quinoline moiety involves C‐atoms 5a and 10 of the benzodiazepine compounds and

c) the hydroxyl group in position 3 of the quinoline ring is derived directly from the epoxide oxygen.

In Scheme 1 a possible reaction sequence for the transformation is given.
Comments on the Kinetics and Mechanism of Yeast Hexokinase Action
Authors: Fromm H. J.
Published: March 2005

The mechanism of action of yeast hexokinase was reinvestigated in light of a recent report in which it was suggested that substrates add to the enzyme in an ordered manner with the binding of glucose required to precede the binding of ATP. Initial velocity experiments were undertaken with AMP which is a competitive inhibitor for ATP, and with the product inhibitor glucose‐6‐phosphate. The results of these studies serve to exclude the ordered mechanism for yeast hexokinase from consideration. A discussion is presented in an attempt to show that yeast hexokinase may react in a random fashion with its substrates before forming a ternary complex of enzyme‐ATP‐glucose.
Stereochemistry of the Enzymatic Conversion of a Δ4‐3‐Oxosteroid into a 3‐Oxo‐5β‐Steroid
Authors: Björkhem I.
Published: March 2005

The synthesis of [4‐3H,4‐14C]7α‐hydroxycholest‐4‐en‐3‐one is described. The transformation of this compound into cholic acid in the bile fistula rat occurred with retention of the tritium label. The position of the tritium label in the isolated cholic acid was established by oxidation at the C‐3 position of the methylated derivative followed by dehydrogenation with selenium dioxide yielding methyl 7α,12α‐dihydroxy‐3‐oxo‐chol‐4‐enoate. This compound retained most of the tritium label. Since selenium dioxide dehydrogenation of a 3‐oxo‐5β‐steroid could be shown to involve a preferential loss of a 4α‐hydrogen, it was concluded that the tritium label in the cholic acid isolated from bile was located in the 4β‐position. The enzymatic reduction of the Δ4 double bond in 7α‐hydroxycholest‐4‐en‐3‐one in its conversion into cholic acid thus involves a trans diaxial addition of hydrogens.
Beziehung zwischen Lipolyse, Gluconeogenese und Insulinsekretion nach Cortisolgabe an Ratten
Authors: Herrmann J. , Staib W.
Published: March 2005

Plasma concentrations of free fatty acids, free glycerin, glucose, immunologically measurable insulin and insulin‐like activity have been studied 0.5–6 h after oral application of 5 mg cortisol/100 g body weight to 24 h starved intact rats.

Free fatty acids decrease significantly within 30 min in comparison with controls and then rise up to a maximum after 4 h; the 4 h value was not statistically different from the controls.

In contrast to control animals blood sugar already shows a significant rise at 2 h. Concentrations of free fatty acids and free glycerin showed no evidence for a cortisol stimulated lipolysis. Therefore and in connection with some theoretical considerations it seems rather improbable that lipolysis products are of any essential importance either as substrates or as regulators in cortisol‐dependent gluconeogenesis.

Immunologically measurable insulin decreases within 1 h after cortisol application (P < 0.05) and reaches a minimum after 2 h which differs from controls with P < 0.001. Insulin‐like activity measured at epididymical adipose tissue showed a similar course with a decrease at 2 h followed by a rise up to the 6 h value.

A significant decrease of plasma insulin concentration could also be found in cortisol treated adrenalectomized rats.

After hypophysectomy an effect of cortisol on plasma insulin concentration could no longer be detected.

Experiments with the isolated perfused rat pancreas show that after glucose stimulation, cortisol does not significantly influence either basal insulin secretion or insulin release. The decrease of plasma insulin concentration of cortisol treated animals can not be explained by a direct inhibition on the interalveolar cell islets by cortisol.

From the different influence of cortisol on the insulin liberation in intact animals, as well as in adrenalectomized ones on the one hand and in hypophysectomized on the other, it is concluded that the observed cortisol effect is mediated by insulotropic adenohypophysis hormones. This view is supported by the fact that a direct influence of the steroid on Langerhans' cells could be excluded by pancreas perfusion.
Enzymic Epimerization of Sequoyitol to d‐Pinitol in Trifolium incarnatum
Authors: Ruis H. , Hoffmann‐Ostenhof O.
Published: March 2005

The partial purification of an enzyme system from Trifolium incarnatum catalyzing the epimerization of sequoyitol to d‐pinitol and its characterization is described. The reaction proceeds via a keto intermediate, d‐5‐O‐methyl‐2,3,5/4,6‐pentahydroxycyclohexanone, and is catalyzed by two dehydrogenases or one protein with two dehydrogenase actions, of which the first one is NAD‐specific and oxidizes sequoyitol to the keto intermediate, whereas the other has the function to reduce the keto compound to d‐pinitol using NADPH as hydrogen donor. Both dehydrogenase actions as well as the overall epimerization reaction were shown to be reversible.

Although the sequoyitol dehydrogenase activity can be easily destroyed by several means which leave the d‐pinitol dehydrogenase activity intact, no real separation of the two activities has been achieved. The protein or proteins responsible for the two activities have a molecular weight of about 34,000 as determined by the gel filtration method.

The two activities do not only differ in their sensitivity towards inhibitors, their substrate and coenzyme specificities, but also in their stereospecificity with respect to the coenzymes. Whereas the NAD‐specific sequoyitol dehydrogenase activity is B‐specific, the NADP‐specific d‐pinitol dehydrogenase activity shows A‐specificity. On the basis of these findings, a possible mechanism for the epimerization is discussed.
Synthesis and Configurational Assay of Asymmetric Methyl Groups
Authors: Cornforth John W. , Redmond John W. , Eggerer Hermann , Buckel Wolfgang , Gutschow Christine
Published: March 2005

By chemical synthesis starting from phenylacetylene and utilizing stereospecific reactions, two specimens of acetic acid were prepared in which the following conditions were closely approached: (a) some of the methyl groups consisted of one hydrogen, one deuterium, and one tritium atom attached to carbon; (b) all such methyl groups in one specimen had the R configuration, and all such methyl groups in the other specimen had the S configuration, and the correct absolute configuration was assignable to each specimen from the method of synthesis used; (c) all methyl groups containing a tritium atom also contained a deuterium atom.

Each specimen of acetic acid was converted enzymically into acetyl‐coenzyme A and (in the same incubation) this product was condensed with glyoxylic acid on malate synthase to form two specimens of S‐malic acid.

Each specimen of malic acid was incubated with fumarase until the tritium content of the organic acids (fumarate + malate) had ceased to fall.

Malic acid synthesized from R‐acetic acid retained 69% of its carbon‐bound tritium (in fumarate + malate) on incubation with fumarase. Malic acid synthesised from S‐acetic acid retained 31% of its carbon‐bound tritium.

The enzymic assay described in paragraphs 2–4 constitutes a method for determining the absolute configuration of asymmetric methyl groups, provided that a substantial proportion of the tritium in the specimen examined is present in such groups.

The method thus established can be used to study the mechanism of enzymic reactions in which a methylene group is converted stereospecifically into a methyl group. With one logical proviso–that it is necessary to known whether protium or deuterium is preferentially removed by the enzyme from methyl groups also containing tritium–the method is also applicable to enzymic reactions in which a methyl group is converted stereospecifically into a methylene group.

Subject to the above proviso, it is indicated that on malate synthase the removal of hydrogen and the attachment of the glyoxylate residue occurs with inversion of configuration.
Complexes of Liver Alcohol Dehydrogenase
Authors: Reynolds C. H. , Morris D. L. , Mckinley‐Mckee J. S.
Published: March 2005

The Michaelis‐Menten‐type kinetics of inactivation of horse‐liver alcohol dehydrogenase with iodoacetate have been used to study the binding of a number of known ligands. Adenine nucleotides protect competitively with iodoacetate as do halides and aromatic acids. Uncharged chelating‐agents (orthophenanthroline, and 2,2′‐bipyridyl) protect non‐competitively. Uncharged monodentate ligands (e. g. imidazole) stimulate inactivation.

The effect of imidazole on the binding of various ligands at pH 7.4 and at pH 10 was studied. Multiple inhibition analysis was used to examine the mutual interaction of pairs of substances which protected the enzyme from iodoacetate. ADP‐ribose and decanoate were mutually competitive, while AMP and orthophenanthroline were non‐competitive. Increasing the ionic strength was found to affect differently the mutual interactions of pairs of anions, as well as to weaken their binding to the enzyme.

In some cases, kinetic studies of reduction of NAD+ were used to complement the inactivation studies.

It is concluded that, in the coenzyme, the phosphate group nearer to adenosine is important for binding. Most, but not all, of the ligand‐ligand interactions can be explained by steric or electrostatic effects; it is tentatively suggested that conformational changes may also be involved. Precise interpretation, however, is complicated: there are two different types of zine in the enzyme, and anions might bind to either, or neither.
Thermochemistry of Lysozyme‐Inhibitor Binding
Authors: Bjurulf Carin , Wadsö Ingemar , Laynez José
Published: March 2005

A microcalorimetric method has been used to study the binding of saccharide inhibitors to lysozyme in aqueous solution at pH 5 and 25°. Inhibitors studied were N‐acetyl‐d‐glucosamine, di‐N‐acetyl‐d‐glucosamine and tri‐N‐acetyl‐d‐glucosamine. From the results of the calorimetric measurements Δ G°, Δ H° and Δ S° for the binding processes were calculated. Derived data have been compared with corresponding values obtained by various equilibrium measurements.
Isolation and Characterisation of the Peptides Derived from the α1‐Chain of Calf Skin Collagen after Hydroxylamine Cleavage
Authors: Heidemann E. , Heinrich W.
Published: March 2005

The α1‐chain of acid soluble calf skin collagen was cleaved with hydroxylamine and the resulting peptides were separated and purified by ion exchange and molecular sieve chromatography.

Four fragments were isolated, representing molecular weights of 20 000, 31 000, 45 000, and 48 000; also intact or probably slightly modified α1‐chains could be found. The peptides were renatured and precipitated as long spacing segment fragments using ATP; their order in the intact molecule was determined, using the electron microscopy, by their cross‐striation patterns.

The different fragments are derived from the B‐end of the α1‐chain, only the shortest peptide derives from a middle region of the molecule. The content of the hydroxamic acids of the fragments varies from 0.59 to 1.18, the modified α1‐chain shows 2 groups of hydroxamic acid.

In four of the five isolated fractions glycine could be determined as N‐terminal.
The Effect of Triton X‐100 on the Respiratory Chain Enzymes of a Heart‐Muscle Preparation
Authors: Soltysiak Dorota , Kaniuga Zbigniew
Published: March 2005

The effect of non‐ionic detergent Triton X‐100 on the respiratory chain enzymes of a heartmuscle preparation was compared with that of deoxycholate.

The efficiency of the action of the detergents on the activity of membrane‐bound enzyme depends on both the detergent/protein ratio and the concentration of protein in the reaction mixture.

No essential differences both in the site and the degree of inhibition or activation of enzymic activities were observed due to the action of Triton X‐100 or deoxycholate when a suitable detergent/protein ratio was applied.

The usefulnes of non‐ionic detergent Triton X‐100 for the studies on several membrane‐bound enzymes is discussed.
Characterization of Neurospora crassa Mitochondria Prepared with a Grind‐Mill
Authors: Weiss Hanns , von Jagow Gebhard , Klingenberg Martin , Bücher Theodor
Published: March 2005

Mitochondria can be isolated rapidly from Neurospora crassa with high yields by disrupting the hyphae suspension between the two grinding wheels of a simple mill.

The isolated mitochondria respire with pyruvate + malate, succinate, and with NADH and NADPH in high rates and well coupled to oxidative phosphorylation.

Three phosphorylation steps are involved in the oxidation of pyruvate and two in the oxidation of succinate, NADH and NADPH.

Rotenone, antimycin and KCN inhibit the electron flow at the known steps.

The cytochrome composition pattern is characterized by a particularly high content of cytochrome c.

Evidence is given for the existence of two NADH‐dehydrogenases for the oxidation of exogenous and endogenous NADH with different localizations on the inner mitochondrial membrane.

On the basis of these properties, mitochondria from Neurospora crassa can be looked upon in analogy to mitochondria from yeast, particularly from Torulopsis utilis.
Structure primaire de la caséine αS1 bovine
Authors: Grosclaude François , Mercier Jean‐Claude , Ribadeau‐Dumas Bruno
Published: March 2005

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αS1‐casein, a single‐chain phosphoprotein, formerly reported to have arginine as an NH2‐terminal, and Leu‐TrpOH as COOH‐terminal residues.

Tryptic digestion of maleyl αS1‐casein (genetic variant B), in theory limited to arginyl bonds, yields eight peptidic fragments, Tm 1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The amino acid composition and the phosphate content of these fragments were determined.

The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. thus, six fragments only–Tm1,2,4,5,6 and 7–arise from the specific cleavage of the αS1‐casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2‐terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to represent respectively the NH2‐ and COOH‐terminal parts of the αS1‐casein chain.

These peptidic fragments–except Tm4 which is devoid of lysine–were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established.

As a control, the tryptic peptides of αS1‐casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride.

In summary, this first step of our work gives: (a) the composition of all the bovine αS1‐casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain; (c) confirmation that the molecular weight of the bovine αS1‐casein monomer is approximately 23 600.
Structure primaire de la caséine αS1 bovine
Authors: Mercier Jean‐Claude , Grosclaude François , Ribadeau‐Dumas Bruno
Published: March 2005

A previous paper described the amino acid composition of the fragments resulting from selective cleavage of the bovine αS1‐casein polypeptide chain at arginyl bonds after blocking the lysyl residues by maleic anhydride.

The fragment Tm2, which is 48 residues long, represents the COOH‐terminal part of the αS1‐casein chain, according to the following observations: (a) it is devoid of arginine; (b) only the peptide Tm2 is coloured by the Ehrlich reagent specific for Tryptophane; (c) the two amino acids released by carboxypeptidase A (successively Tryptophane and Leucine) are those of the COOH‐terminal end of αS1‐casein.

The complete sequence of this large COOH‐terminal peptide obtained from αS1‐casein C was determined by classical methods, using endopeptidases to produce smaller peptides, then exopeptidases and the Edman sequential degradation procedure for establishing the amino acid sequence of these peptides.

A part of this work has been published in a previous communication reporting the location, at the eighth position from the COOH‐terminal end, of the substitution Gly/Glu between the genetic variants αS1C and αS1B.
Chloride‐Hydroxide Exchange across Mitochondrial, Erythrocyte and Artificial Lipid Membranes Mediated by Trialkyl‐ and Triphenyltin Compounds
Authors: Selwyn Michael J. , Dawson Alan P. , Stockdale Martin , Gains Nigel
Published: March 2005

Studies of the effects of trimethyltin, tripropyltin, tributyltin and triphenyltin chlorides on the permeability of the inner mitochondrial membrane by the osmotic swelling technique in NaCl and NH4Cl media show that these compounds mediate transport of chloride ions. Studies in KCl media show that these compounds mediate a strictly coupled chloride‐hydroxide antiporter transport. This exchange allows rapid titration of mitochondrial contents by pulses of HCl. Similar results on erythrocytes and smectic mesophases indicate that this is a direct action of the tin compounds and is not due to modification of a pre‐existing anion carrier in the membrane.
The Effects of Polyhydric and Monohydric Alcohols on the Heat Induced Reversible Denaturation of Chymotrypsinogen A
Authors: Gerlsma Sicco Y.
Published: March 2005

The reversible thermal denaturation of chymotrypsinogen A was investigated in aqueous solutions of mono‐, di‐ and polyvalent alcohols.

Sorbitol and erythritol show a stabilizing effect, whereas the di‐ and monovalent alcohols are destabilizing. Divalent alcohols have a less destabilizing influence than monovalent alcohols when equimolecular proportions of ‐CH2‐ and / or –CH3 groups are present.

Stabilizing effects of the polyalchols can be explained in terms of a lessened H bond rupturing capacity of the medium.

The destabilizing by di‐ and movovalent alcohols is attributed to their hydrophobic interaction with the protein, which increases with the number of ‐CH2‐ and, or ‐CH3 groups and decreases with the number of ‐OH groups.

Chymotrypsinogen A is more destabilized by hydrophobic alcohols than ribonuclease.
Interaction of Proteins with Sulfide
Authors: Cavallini Doriano , Federici Giorgio , Barboni Erminia
Published: March 2005

The addition of sodium sulfide to disulfide‐containing proteins dissolved in 0.01 N NaOH produces an absorption in the range of 320–350 nm similar to that exhibited by cystine or cystamine treated under the same conditions. Proteins free of cystine and cysteine and proteins containing disulfide bonds which have been reduced with sodium borohydride do not produce this absorbance. Urea and guanidine at 6 M concentration increase the rate of production of the absorbance while sodium dodecyl sulfate at concentration of 0.02 M has an inhibitory effect both on the rate and the extent of the absorbance. The effect of pH, concentration of reagents and of the additions of cyanide and hypotaurine has been studied. The 320–350 nm absorbance as been related to the production of persulfide groups (R‐SSH) as a result of the cleavage of disulfide bonds by sulfide. Attempts to prepare a purified model protein containing persulfide groups have failed owing to the instability of the persulfide group in conditions far from those used for its preparation.
Structural Investigations on the 2‐Keto‐3‐Deoxyoctonate Region of Lipopolysaccharides
Authors: Dröge Wulf , Lehmann Volker , Lüderitz Otto , Westphal Otto
Published: March 2005

Mild acid hydrolysis of the glycolipid derived from a Salmonella Rd2 mutant led to the liberation of three major split products which, after separation by electrophoresis, were identified as free 2‐keto‐3‐deoxyoctonate (KDO), KDO‐7‐phosphorylethanolamine, and l‐glycero‐α‐d‐mannoheptosyl‐1,5‐KDO. By the use of various methods of KDO determinations, and by periodate oxidation performed on the glycolipid, results were obtained which indicate that the bridge between the polysaccharide portion of lipopolysaccharides and lipid A is formed by a branched KDO trisaccharide whose KDO side chain is randomly substituted by phosphorylethanolamine according to the formula —Hep‐1,5‐KDO‐KDO‐lipid A | KDO‐(phosphorylethanolamine) Analogous results were obtained with P‐lipopolysaccharides of other Salmonella mutants.
Regulation of Histidine Uptake by Specific Feedback Inhibition of Two Histidine Permeases in Saccharomyces cerevisiae
Authors: Crabeel Marjolaine , Grenson Marcelle
Published: March 2005

Although a large fraction of the histidine accumulated from the medium remains free and intact in Saccharomyces cerevisiae, it is not exchangeable with external histidine. Hence, the steady state concentration level of histidine is not determined by a balance between rates of inflow and outflow.

Preloading the cells with histidine results in a rapid inhibition of histidine uptake, whereas preloading with other amino acids has no effect on histidine uptake.

The inhibition by internal histidine affects the activity, and not the synthesis, of two specific histidine permeases. The histidine permease with high affinity for histidine is much more sensitive to this inhibition than the second histidine permease.

A mutant (his‐p1) specifically affected in histidine uptake was isolated.

Criteria for demonstrating that a mutation or a feedback control directly affect an uptake system are discussed.

It is concluded that the his‐p1 mutant is histidine‐permease‐less, and that the activity of the histidine permeases are regulated by specific feedback inhibition.
Solubilisation de l'acétylcholinestérase des organes électriques de gymnote
Authors: Massoulié Jean , Rieger François , Tsuji Shigeru
Published: March 2005

Further evidence is presented in this paper concerning the occurence of three different molecular species of acetylcholinesterase in the electric eel's electric organ. These species, A, C, and D, differ in their sedimentation coefficients (8.5 S, 14.2 S, and 18.4 S, respectively). We show that the whole of the tissue acetylcholinesterase activity can be solubilized by repeated homogenisation, in the form of A, C, and D.

Homogenisations under different conditions of ionic strength, or after prolonged autolysis yield closely similar results: the ratios of A, C, and D are remarkably constant. Moreover ammonium sulfate precipitation does not affect these species. This raises the question of their relationship and significance. All three species are recognized by antibodies obtained from rabbit immunized against the purified enzyme.

We have also studied the solubilization of acetylcholinesterase by trypsin in electric tissue slices. This is very effective and directly yields species B. This species, which is homogeneous in sedimentation, but might contain a variety of slightly different protein molecules, can also be obtained by partial tryptic digestion of A, C, and D. The sedimentation coefficient of species B is very close to that of purified acetylcholinesterase and we have previously suggested that the two kinds of molecules may be similar. Even though this is probably correct, we have been able to detect a significant difference (0.8 S unit) in the sedimentation coefficients.
Regulation of the Enzymes of the β‐Ketoadipate Pathway in Moraxella
Authors: Tresguerres M. Elena F. , DE Torrontegui Gertrudis , Ccánovas José L. , Ingledew W. Michael
Published: March 2005

The ability to oxidize quinate is elicited in Acinetobacter calco‐aceticus not only by growth on quinate but also by growth on protocatechuate or its precursors such as shikimate and p‐hydroxybenzoate. Accordingly the synthesis of a quinate dependent dehydrogenase was found to be induced by protocatechuate. Moreover, this enzyme seems to belong to the same coordinate block of enzymes responsible for the conversion of shikimate to β‐ketoadipyl‐CoA.

Analogies between quinate and shikimate oxidation in A. calco‐aceticus have been substantiated by showing that both quinate dehydrogenase and shikimate dehydrogenase activities rely on a single enzyme.

Several auxotrophs that have lost the ability to utilize both quinate and shikimate as sole source of carbon and energy have been isolated. Particularly relevant for this study are two of these mutants in which the dehydrogenase is severely impaired and correspondingly their ability to oxidize the mentioned hydroaromatic compounds is substantially reduced. The properties of these auxotrophs and their revertants confirm the key role of the dehydrogenase in the dissimilation of both quinate and shikimate. The rationale of the control of this enzyme by protocatechuate is discussed.
ω‐Oxidation of Branched‐Chain Fatty Acids in Rat Liver Homogenates
Authors: Björkhem Ingemar , Danielsson Henry
Published: March 2005

The synthesis of tritium‐labeled 14‐methylpentadecanoic acid, 15‐methylhexadecanoic acid and 16‐methylheptadecanoic acid is described. These acids were found to be oxidized into a number of water‐soluble and ether‐soluble products by the 800×g supernatant fluid of rat liver homogenate. In the presence of mitochondrial or microsomal fraction fortified with NADPH, the iso‐fatty acids were converted mainly into ω‐oxidized products. Addition of boiled 100 000×g supernatant fluid or NAD increased the yield of ω‐oxidized products. Gas‐chromatographic and mass‐spectrometric analyses of the products formed showed that the main metabolites were the ω‐hydroxy iso‐fatty acid and the corresponding dicarboxylic acid.
Leucyl‐tRNA Synthetase
Authors: Rouget Pierre , Chapeville François
Published: March 2005

Two forms of leucyl‐tRNA synthetase of Escherichia coli B were separated and purified about 850‐fold to apparent homogeneity. Their molecular weights were identical, 104000, as determined by Sephadex filtration and polyacrylamide electrophoresis.

One of these forms catalyzed the leucine‐dependent ATP‐PPi exchange reaction but was unable to transfer leucine to tRNA even though it could form a complex with tRNALeu. The other form was active for both ATP‐PPi exchange and charge of tRNA.

Evidence was obtained for the interconversion between these two forms of leucyl‐tRNA synthetase and for the existence in the E. coli 105000Xg supernatant of a factor stimulating this interconversion; during the incubation of one of these forms with the supernatant, the other form appeared and an apparent equilibrium between the two forms was observed.
Reduction in Immunological Manifestations of 6‐Aminopenicillanic Acid by Treatment with Water‐Insoluble Pronase
Authors: Shaltiel Shmuel , Mizrahi Rachel , Sela Michael , Stupp Yehudit
Published: March 2005

Penicilloylated proteins which are found as an impurity in 6‐aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the potent polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carried out in 6‐aminopenicillanic acid preparations, since even a large excess of 6‐aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme.

In order to avoid the contamination of 6‐aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process.
ATP: Glutamine Synthetase Adenylytransferase from Escherichia coli B
Authors: Ebner Eberhard , Wolf Dieter , Gancedo Carlos , Elsässer Sigrid , Holzer Helmut
Published: March 2005

Glutamine synthetase adenylytransferase was purified 300‐fold from Escherichia coli B grown on minimal medium. The purified enzyme was found to be homogeneous in the ultracentrifuge, but by electrophoresis on polyacrylamide gel two enzymatically active protein components were separated, that differed from batch to batch in their relative proportions.

The apparent Km values of the various substrates and activators of the adenylylation of glutamine synthetase were determined.

The adenylylating enzyme requires Mg2+ or Mn2+ and ATP for activity. Several other divalent cations and nucleotides were tested and found to be ineffective. In the presence of glutamine the rate of the reaction decreases at ATP concentrations greater than 1 mM. Addition of ADP, CTP, UTP and ITP to the standard assay mixture, containing ATP results likewise in a decrease of the rate of the reaction. Various intermediates of glycolytic pathway and Krebs cycle inhibit the adenylylation reaction. The optimum pH for adenylylation is about 7.6. The activation energy for the adenylylation was estimated from rate measurements. It is 13 kcal/mole in the temperature range from 5° to 32°. Gel filtration on Sephadex G‐150 indicates an average molecular weight of 145000 daltons. On storage of the enzyme in solution inactive aggregates of higher molecular weight are formed. Bovine serum albumin prevents inactivation and aggregation.