bluuebunny/crossref_metadata_2025 huggingface dataset:
Physico‐chemical Properties of Native and Recombined Calf Thymus Nucleohistones
Authors: Fredericq E. , Houssier C.
Published: March 2005

In the preparation of calf thymus deoxyribonucleohistone a soluble component is formed when the time of storage of the glands before extraction is too high, when high speeds of homogenization are used or when dilute solutions are too strongly stirred. Under special experimental conditions, a soluble component can be obtained which is slightly degraded in comparison with the native gel‐forming deoxyribonucleohistone, its protein content and its molecular weight being a little lower; electric birefringence and dichroism are however similar in both cases.

The dissociation of deoxyribonucleohistone in concentrated salt solutions was followed by centrifugation at 100,000×g under various conditions. A progressive lowering of the protein content was found in the pellet, when the concentration varies from 0.4 to 3 M NaCl; a minimum value of 0.05 was found for the protein/DNA ratio after three centrifugations in 4 M LiCl. The analysis of the residual proteins which are undissociable from DNA shows a predominance of acidic over basic amino‐acids.

Deoxyribonucleohistone dissociated in salt solutions and recombined is similar to native deoxyribonucleohistone, as regards the rigidity, the intrinsic viscosity, the melting temperature and the electro‐optical parameters. Combinations of histone and DNA display different properties.

Deoxyribonucleohistone with various protein contents were prepared by centrifugation and their characteristics compared with those of native deoxyribonucleohistone. It is only for low protein/DNA ratios that most of the properties are significantly altered.

The formation of gels requires a high histone content and the presence of specific proteins. The significance of gel appearance and the necessity for quantitative measurements of the modulus of rigidity have been outlined.
Ion Transport in Liver Mitochondria
Authors: Rossi C. , Azzone G. F. , Azzi A.
Published: March 2005

The H+/Ca++ ratio during the aerobic uptake of Ca++ by rat liver mitochondria, oxidizing endogenous substrates or succinate, increased with the increase of the Ca++/protein ratio. At high protein concentrations an apparent “reuptake” of the H+ ejected was observed. Mitochondrial aging also resulted in a decrease of the H+/Ca++ ratio.

Succinate caused a decrease of the H+ ejection. The effect of succinate was dependent on the pH and on the concentration of Ca++. A binding of succinate to the mitochondria was observed parallel to the decrease of the H+/Ca++ ratio from 2 to 1.

Addition of Ca++ caused a release of K+ which was slow in fresh mitochondria and rapid in valinomycin treated mitochondria. The K+/Ca++ ratio was 2 in both cases.

A H+/Ca++ ratio of about 2 was usually observed when the aerobic uptake of Ca++ was started either by the addition of mitochondria to a Ca++ containing medium or by the addition of succinate to Ca++‐and rotenone‐treated mitochondria.
Levels of Glycolytic Intermediates in the Musculature of the Chick during Embryonic and Post‐Embryonic Development
Authors: Arese P. , Rinaudo M. T. , Bosia A.
Published: March 2005

Levels of glycogen and of glycolytic intermediates were assayed in quickly frozen embryonic mesodermic tissue (decapitated and eviscerated chick embryos) at the 5th and 7th developmental day and in the hind limb musculature in subsequent pre‐ and post‐natal stages. Serial determination of dry weight values during embryonic development allows conversion of the levels which are reported on a wet weight basis.

While glycogen gradually increases from the 9th day onwards reaching about 1/3 of the adult amount on hatching, no substantial differences between early embryonic and adult levels were noted in the case of other intermediates, such as glucose, dihydroxyacetone‐phosphate, glyceradehyde‐3‐phosphate, 3‐ and 2‐phosphoglycerate, phosphoenolpyruvate, pyruvate and lactate. Glucose‐1‐phosphate, fructose‐6‐phosphate, glucose‐6‐phosphate and fructose‐1,6‐diphosphate were low during most of the embryonic development, attaining levels close to the adult ones only on hatching.

In order to study the flow rates, the equilibrium situation at each single step and the possible control points along the glycolytic pathway, the tissue was stressed by brief periods of ischaemia or by electrical stimulation at different stages of development.

Glycolytic flow rates showed a progressive increase up to hatching, where a lactate accumulation of 1.5 μmolc/min/g wet weight was measured. The maximum rate of 12.9 μmoles/min/g wet weight found in electrically stimulated muscle of hatching chicks, is well below the adult value of 52.7 μmoles/min/g wet weight.

The approximate stoichiometric correspondence between glucose + glycogen breakdown and lactate formation, found to exist in the early and late embryonic stages, is absent on the 7th and incomplete on the 11th day, where the sharply reduced gluco‐ and glycogenolysis cannot account for the measured glycolytic rate. The existence of a vicarious anomalous glycolysis, supplying pyruvate and lactate by transamination or by other unknown mechanisms, is possible at these two stages.

The constancy of mass‐action ratios under altered glycolytic fluxes and the comparison between the ratios in vivo and the thermodynamic equilibria show that phosphoglucomutase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, phosphoglycerate mutase and enolase were maintained either at equilibrium or fairly close to equilibrium. Owing to the probable compartmentation of ATP and ADP in muscle, no calculation of kinase‐catalyzed reactions was attempted.

Activation of glycolysis could not be observed on electrical stimulation of embryonic muscle at the 14th day, as the constancy of such indicator metabolites as glucose‐6‐phosphate, fructose‐1,6‐diphosphate and lactate demonstrates. An activation at the phosphorylase and phosphofructokinase steps has been indirectly shown on hatching.
Synthesis, Characterization and Enzymatic Properties of Poly‐l‐Lysyl Ribonuclease
Authors: Frensdorff A. , Sela M.
Published: March 2005

Poly‐l‐lysyl RNase was prepared by the reaction of RNase with ɛ, N‐trifluoroacetyl‐α, N‐carboxy‐l‐lysine anhydride, either in dioxane‐aqueous phosphate buffer or in dimethylformamide in the absence of phosphate. The trifluoroacetyl group was subsequently removed from poly‐trifluoroacetyllysy RNase by 1.0 M aqueous piperidine. Derivatives prepared in aqueous media and enriched with up to 70 lysine residues, and derivatives prepared in dimethylformamide enriched with up to 270 lysine residues, were soluble and retained some activity. This activity could be completely recovered in all preparations after reduction of the disulfide bonds, followed by reoxidation. Approximately four phenolic hydroxyl groups were found, by spectrophoto‐metric titration, to ionise normally in polylysyl RNase, as opposed to three in the native enzyme.

The enzymic activity of polytrifluoroacetyllysyl RNase, both on RNA and on cytidine 2′,3′‐cyclic phosphate, under various conditions, was essentially identical to that of polyalanyl RNase. Polylysyl RNase, in which the amino groups of the attached lysine peptides are unblocked and protonated, is active on RNA as well as on cytidine 2′,3′‐cyclic phosphate, but optimal activity is obtained under conditions different from those of the native enzyme. Thus polylysyl RNase displays maximal activity on RNA at ionic strength 0.6–0.7. The optimum pH for activity depends on ionic strength and is shifted at low ionic strength in the same direction as that of the isoelectric point of the modified enzyme for both RNA and cytidine cyclic phosphate. The base specificity of the modified enzyme was not significantly modified.

The changes in ionic strength and pH dependence of activity are related directly to the lysine peptides attached to the enzyme and can be reversed by either reblocking the free amino groups or by removing the peptides with trypsin. Kinetic analyses suggest that the changes in activity are a result of modified substrate binding.
Regulation of the Enzymes of the β‐Ketoadipate Pathway in Moraxella calcoacetica
Authors: Cánovas J. L. , Stanier R. Y.
Published: March 2005

The inducible enzymes responsible for the dissimilation of aromatic compounds through the β‐ketoadipate pathway by Moraxella calcoacetica are subject to a high degree of coordinate control. The five enzymes which catalyze the conversion of protocatechuate to β‐ketoadipyl‐CoA are coordinately synthesized, the probable inducer being protocatechuate. In the conversion of catechol to β‐ketoadipyl‐CoA, there are two separate inductive events, though both are mediated by one metabolite‐inducer, cis,cis‐muconate. One is the induction of catechol oxygenase; the other is the coordinate induction of the four enzymes that catalyze conversion of cic,cis‐muconate to β‐ketoadipyl‐CoA.

Although the catechol and protocatechuate branches of the β‐ketoadipate pathway converge metabolically with the formation of β‐ketoadipate enol‐lactone, the two step‐reactions required for the conversion of this intermediate to β‐ketoadipyl‐CoA are mediated by two isofunctional sets of inducible enzymes, one controlled coordinately with enzymes mediating steps specific to the catechol branch, the other coordinately with enzymes mediating steps specific to the protocatechuate branch.

The differing control mechanisms which govern synthesis of the enzymes of the β‐ketoadipate pathway in Moraxella calcoacetica and in Pseudomonas putida are compared and discussed.
Radical Transfer Reactions and Chemical Protection in the Dry State. Electron Spin Resonance Studies on Irradiated Mixtures of Ribonuclease and Glycyl‐Glycine
Authors: Copeland E. S. , Sanner T. , Pihl A.
Published: March 2005

Molecular mixtures containing different proportions of ribonuclease and glycyl‐glycine were irradiated with X‐rays at 77° K and the electron spin resonance spectra were observed at this temperature and after heat‐treatment for 10 minutes at 60°.

In mixtures freeze‐dried from pH 8 the number of unpaired spins observed at 77° K corresponded to the weighted average of the yields for the components. In contrast, in mixtures freeze‐dried from solutions at pH 4.4 the number of unpaired spins was much higher than expected. The qualitative spectra indicated that these “excess radicals” were located on glycyl‐glycine.

The results obtained after heat‐treatment indicated that in the samples freeze‐dried from pH 8 a considerable transfer of spins occurred from RNase to glycyl‐glycine during the heat‐treatments, whereas in the samples freeze‐dried from pH 4.4 little or no additional transfer seemed to occur.

The results indicate that the number of “excess radicals” at 77°K, as well as the extent of interaction in the mixtures which occurred upon heat‐treatment, depended on the state of the dissociable groups of the constituents. The finding of “excess radicals” at 77° K is tentatively attributed to the action of H atoms ejected from RNase. These H˙ atoms are assumed to interact with glycyl‐glycine to form stable radicals, while in the absence of glycyl‐glycine they are assumed to disappear primarily in combination reactions.
An Evaluation of the Mitchell Hypothesis of Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation
Authors: Slater E. C.
Published: March 2005

The Mitchell hypothesis of chemiosmotic coupling in oxidative phosphorylation is examined in the light of experimental data on oxidative phosphorylation at present available. The following objections are brought against the theory:

Data on the magnitude of the phosphate potential against which ATP can be synthesized by the respiratory chain require, on the basis of chemiosmotic coupling, very effective proton and/or cation extrusion and/or anion uptake by the mitochondria. Experimental evidence for this is lacking. Indeed it has been shown by Chance and Mela that mitochondria in the controlled state do not extrude protons.

Although reversible ATP‐driven proton (chloroplasts) or cation (mitochondria, erythrocytes) pumps have been demonstrated, this is insufficient evidence for the postulate that the establishment of a proton gradient is the primary energy‐conserving event of the chloroplast or mitochondrial respiratory chain. Indeed, it is general experience that cations are required for the extrusion of protons by mitochondria.

The stoicheiometry of proton extrusion by mitochondria and of proton uptake by chloroplasts, and the kinetics of the latter process are also difficult to reconcile with the chemiosmotic hypothesis.

Further experimental evidence is presented confirming that, under the conditions of the oxygen‐pulse experiments of Mitchell and Moyle, the extrusion of H+ is not associated with the oxidation of mitochondrial NADH.

The respiratory chain included in the chemiosmotic hypothesis is difficult to reconcile with our present knowledge of the chain.

It is concluded that the chemiosmotic theory, in its present form, is untenable.
On the Mechanism of Na+‐and K+‐Stimulated Hydrolysis of Adenosine Triphosphate
Authors: Schoner W. , Von Ilberg C. , Kramer R. , Seubert W.
Published: March 2005

The purification and properties of a Na+‐ and K+‐activated ATPase from ox brain is described. The enzyme preparation is characterized by a high purity and a low content (<1%) of Mg++‐stimulated ATPase.
Differences between the Chemical Structures of Duck and Hen Egg‐white Lysozymes
Authors: Jollès J. , Hermann J. , Niemann B. , Jollès P.
Published: March 2005

Histidine‐free duck egg‐white lysozyme III and hen egg‐white lysozyme have similar but not identical structures. some structural differences are reported.

The N‐ and C‐terminal sequences are different.

The unique histidine and two phenylalanine residues of hen lysozyme are replaced by a leucine and two tyrosine residues, respectively.

The more basic character of duck lysozyme III is due to additional arginine residues.

Duck egg‐white lysozymes II and III have not entirely identical chemical structures.
Studies on Phytosterol Biosynthesis: the Sterols of Larix decidua Leaves
Authors: Goad L. J. , Goodwin T. W.
Published: March 2005

The sterols of Larix decidua leaves consist of β‐sitosterol, campesterol, cycloartenol, 24‐methylene cycloartanol, cycloeucalenol, 24‐methylene lophenol, 24‐ethylidene lophenol, together with a number of unidentified 4α‐methyl sterols. The pattern of incorporation of [2‐14C]mevalonic acid into these compounds has been examined.
The Effect of Sulfhydryl Reagents on Ribosome Activity
Authors: Traut R. R. , Haeeni A. L.
Published: March 2005

Ribosomes from Escherichia coli were incubated with sulfhydryl reagents and then after removal of the excess reagent assayed for polyuridylic acid dependent polyphenylalanine synthesis in the absence of both sulfhydryl reagents and thiols. Incubation with N‐ethylmaleimide, dithiobis‐(2‐nitrobenzoic acid) and p‐chloromercuribenzoic acid resulted in loss of activity between 30 and 70% in different ribosome preparations. Similar inactivation was observed in the polylysine assay system and in polyuridylic acid directed leucine incorporation (miscoding). Inactivation by dithiobis‐(2‐nitrobenzoic acid) was reversed by incubating the ribosomes with a thiol. The extent of inactivation was independent of whether the ribosomes were treated with the sulfhydryl reagent in the associated (70 S) or dissociated (50 S + 30 S) form. The sedimentation coefficients of the treated ribosomes and of the subunits prepared from them were not altered. The treated ribosomes were in the associated (70 S) form under the conditions of polyphenylalanine synthesis. No inhibition of the binding of polyuridylie acid was found. Inactivation occurred when the molar ratio of sulfhydryl reagent to 70 S ribosome was only 2 or 3, although the calculated half cystine content of the 70 S ribosome is 45 residues. The 30 S ribosome was the principle site of inhibition. The results indicate that at least some ribosomal half cystine is present as cysteine and that ribosomal SH groups, as well as supernatant transfer factor SH groups, are important in protein synthesis.
Sequenzanalyse von Oligonucleotiden mit Hilfe von Micrococcus‐Nuclease
Authors: Feldmann H.
Published: March 2005

Micrococcal nuclease has been used in determination of oligonucleotide sequences. In order to study the mode of splitting of this enzyme in more detail, seven oligonucleotides of known sequences were digested under varying conditions.

The splitting patterns obtained from the tetranucleotides CpCpCpG, ApApApG and ApΨpUpI, the pentanucleotides UpCpCpUpG, CpUpUpUpG, and UH2pUH2pApApG, and the heptanucleotide CpApApCpUpUpG were determined by analysis of the degradation products. The digestions were carried out with a crystalline sample of the enzyme. The results agree with and extend earlier observations with non‐crystalline fractions of micrococcal nuclease.

A comparison of the splitting patterns leads to some generalizations: from all oligonucleotides investigated so far, the 3′‐terminal dinucleoside phosphate and the 5′‐terminal dinucleoside diphosphate were released under appropriate conditions. Tetranucleotides were split preferentially in the middle, pentanucleotides as to give mainly the dinucleotides and the middle mononucleotides. The digestion of longer oligonucleotides gave more complicated splitting patterns. The information obtained from the analyses was sufficient to reconstruct the nucleotide sequences in all cases.

Micrococcal nuclease digestion as described here should be a generally applicable method for the sequence determination of oligonucleotides.
La maltodextrine phosphorylase d' Escherichia coli
Authors: Schwartz M. , Hofnung M.
Published: March 2005

Purification of the polysaccharide phosphorylase which is induced by maltose in Escherichia coli is described. A forty five‐fold purification is achieved by heating, two ammonium sulfate precipitations and chromatography on DEAE‐cellulose. Purified preparations are essentially homogenous on DEAE‐cellulose, by electrophoresis on polyacrylamide gels and by sedimentation in the ultracentrifuge.

The sedimentation coefficient s25,w of the enzyme is equal to 8 Svedberg units by sucrose gradient centrifugation and by ultracentrifugation at various protein concentations. The diffusion constant D25,w determined by the synthetic boundary technique, is 5,5 × 10−7 cm2× sec−1. The molecular weight which is derived from s25,w and D25,w (130,000) is in good agreement with that obtained by sedimentation equilibrium (140,000).

The absorption spectrum of the purified enzyme has a shoulder around 330 mμ, which suggests the presence of bound pyridoxal phosphate as has also been found in polysaccharide phosphorylases obtained from various sources.

The coenzyme is further identified in the present study by its high reactivity towards phenylhydrazine in perchloric acid supernatants of the enzyme. Based on the assumption that the specific molecular extinction of bound PLP is similar in all polysaccharide phosphorylases, a PLP/protein ratio of 1 mole/147,000 g can be derived from the absorbance of phosphorylase solutions at 333 mμ. The direct estimation of the quantity of PLP released in perchloric acid supernatants gives 1 mole/142,000 g.

These results indicate the presence of 1 mole of PLP per mole of protein and suggest that maltodextrin phosphorylase is a monomeric enzyme.

Enzyme stability, pH optimum and affinity for inorganic phosphate have been studied. Short, linear, α(1 → 4) linked oligoglucosides are very active substrates, whereas polyglucosides of high molecular weight (glycogen, starch) are much less active. The shortest maltodextrin which can readily be phosphorolysed is maltopentaose.

The maltodextrin phosphorylase of Escherichia coli appears to be similar to animal and plant polysaccharide phosphorylases in several of its enzymatic properties. Furthermore, a point of striking similarity is the presence of bound PLP.

The bacterial enzyme has two distinctive properties: one is its specificity for small oligoglucosides and the other is its monomeric structure.
Selfassembly of Defective Particles of the Bacteriophage fr
Authors: Hohn T.
Published: March 2005

The assembly of protein subunits and RNA of the icosahedral, RNA‐containing bacteriophage fr was studied. Conditions were found under which the constituents could be specially reaggregated to form virus‐like particles, a small fraction of which was infective. The RNA seems to be essential for the assembly process.
Ca++‐Dependent Movements of H+ and K+ across the Rat Liver Mitochondrial Membrane
Authors: Carafoli E. , Rossi C. S.
Published: March 2005

During uptake of Ca++ by rat liver mitochondria in the absence of freely permeant anions there is an incomplete exchange of positive charges, since only one H+ is ejected for each Ca++ taken up. The studies reported in this communication deal with the possibility that other cations, namely K+, leave the mitochondria during the active translocation of Ca++. The results show that: (a) when the mitochondrial membrane is intact and thus impermeable to K+, no ejection of K+ occurs; (b) when the mitochondrial membrane is rendered permeable to K+, then K+ is ejected, and partially replaces H+ in exchange with Ca++. The tentative conclusion is offered that two pools of K+ may exist in mitochondria, in two physical states or two different compartments. One is the endogenous K+ of intact mitochondria, which is not accessible to the exchange with Ca++; the other pool includes excess K+ that is taken up in a non‐specific process, or by specific valinomycin‐dependent induction. The latter pool of mitochondrial K+ may replace H+ in the exchange with Ca++.
Mechanism of the Action of Chelating Agents on Proline Hydroxylation and its Incorporation into Collagenous and non‐Collagenous Proteins
Authors: Chvapil M. , Hurych J. , Ehrlichová E. , Tich M.
Published: March 2005

The effect of concentration of some chelating agents on proline hydroxylation and the synthesis of collagenous and non‐collagenous proteins was studied in skin slices of chick embryo. In a group of eight chelating agents a good correlation was found between the degree of inhibition of hydroxylation and the stability constant of ferrous chelates. No correlation with the stability of cupric complexes was found. The failure of 2,9‐dimethyl‐1,10‐phenanthroline to inhibit hydroxylation also suggests participation of Fe2+. The study of the influence of different times of preincubation of the tissue with chelating agents showed that 2,2′‐dipyridyl and 1,10‐phenanthroline inhibit the hydroxylation of proline already within 10 minutes. In addition 2,2′‐dipyridyl and in 1 mM concentration also inhibits after 2 hours of action the incoporation of [14C]proline into collagenous proteins and does not change during a 4 hour incubation the synthesis of non‐collagenous proteins. Phenanthroline, in the same concentration, inhibits already during the first 60 minutes of incubation both the synthesis of collagen as well as of non‐collagenous proteins. Chelating agents which do not penetrate cell membrane are without effect on the reactions studied.

The blockade of hydroxylation is a specific result of the chelation of bivalent iron. Chelating agents influence more easily the incorporation of amino acids into collagenous proteins than synthesis of globular proteins. This effect is considered specific because it is related to the chelating properties of the substances investigated. All other investigated substances without chelating properties inhibit, according to the concentration used, synthesis of both types of protein equally, so that the decrease in hydroxyproline formation is secondary to the inhibition of proline incorporation. Close correlation between the inhibition of protein synthesis and general toxicity of these substances has been found.
Fat Transport in the Locust: The Role of Diglycerides
Authors: Tiezt A.
Published: March 2005

An analysis of the lipids of the fat body of Locusta migratoria showed that approximately 97% are triglycerides and 2–3% diglycerides; free fatty acids and phospholipids were present only in small amounts. In the hemolymph, diglycerides and phospholipids were the major lipid components; free fatty acids were present in very small amounts, triglycerides were absent. The concentration of the diglycerides in the hemolymph was found to increase with the age of the locust and during sustained flight; the phospholipids remained constant. The concentration of the free fatty acids in the tissue and hemolymph was also increased during flight.

When fat body tissue was incubated in vitro with [1‐14C]palmitate, the acid was incorporated into the di‐ and triglycerides of the tissue. The specific activity of the diglycerids was at least 50 times higher than that of the triglycerides.

When prelabeled tissue was transfered into a medium containing hemolymph, diglycerides were released into the medium. The amount of diglyceride released was dependent on the time of incubation and on the amount of hemolymph which was added to the medium. Although the amount of diglycerides which was released, was greater than their concentration in the tissue, the amount of the latter did not change. However, their specific activity was very markedly reduced. When the fat body tissue was prelabeled in vivo and the specific activity of the di‐ and triglycerides was identical, the specific activity of the diglycerides was not reduced during diglyceride release.

When fat body tissue of locusts after flight was incubated with [1‐14C]palmitate, incorporation of the acid into the diglycerides was very markedly reduced. This inhibition was reversed by the addition of glucose to the medium.

When prelabeled tissue of locusts after flight was incubated in hemolymph obtained from the same locusts, diglycerides and free fatty acids were released into the medium.
Comparative Studies of Amino Acid Incorporation in a Cell‐free System from Psychrophilic Pseudomonas sp.412
Authors: Krajewska E. , Szer W.
Published: March 2005

A subcellular amino acid incorporating system has been obtained from the psychrophilic bacterium Pseudomonas sp. 412. The system is active from near 0° to 45°, i. e. about 15° above the maximal growth temperature of the organism. The Mg2+ requirement of the system increases from 7 mM at 2° to 14–18 mM at 45°.

The sedimentation properties and optical density melting profiles of ribosomes from Pseudomonas sp. 412 have been found similar to those of Escherichia coli ribosomes.

The poly U‐induced leucine incorporation is negligible in this system as compared to the E. coli and Bacillus stearothermophilus systems.

The ribosomes and supernatant fractions of Pseudomonas sp. 412 and E. coli are interchangeable at 30–45° in the poly U‐promoted phenylalanine incorporation. At 2–9° only Pseudomonas sp. 412 ribosomes are active with both supernatant fractions. The ability to perform protein synthesis near 0° is related to ribosomal specificity. The E. coli soluble enzymes are active near 0° if combined with the ribosomes from the psychrophilie strain.
C19‐Steroid‐glucuronide in menschlichem Plasma nach intravenöser Injektion von freiem [7α‐3H]‐3β‐Hydroxy‐Δ5‐androstenon‐(17)
Authors: Knapstein P. , Wendlberger F. , Oertel G. W.
Published: March 2005

Free 7α‐[3H]dehydroepiandrosterone (DHEA) was injected intravenously into a 46 year old woman; 10 minutes afterwards the 3H‐labelled glucuronosides of dehydroepiandrosterone, androsterone, etiocholanolone and Δ4‐androstendione could be isolated and identified in peripheral venous plasma; 2% of the conjugated dehydroepiandrosterone were found to be coupled with glucuronic acid and 98% with sulphuric acid.
Études immunochimiques sur les Salmonella
Authors: Nghiem H. O. , Bagdian G. , Staub A. M.
Published: March 2005

In a preceding publication Le Minor and Staub [8] predicted from phagic and immunological studies, that the repeating unit of the group D2 polysaccharide should be:

In this paper the specific polysaccharide was prepared according to Freeman's technique and hydrolysed. High yields of 3 oligosaccharides have been obtained whose analysis gave:

Periodate oxydation of the polysaccharide intact or degraded by slight hydrolysis (N CH3COOH, 4 hours at 100°), confirmed these data and showed that tyvelose is linked to carbon 3 of mannose. No O‐acetyl was found.

Immunological data showed that tyvelose was the immunodominant sugar for both factors (9) and 46. No other analysis of these factors was possible since we did not posses oligosaccharides containing tyvelose. The position of the different factors of Salmonella strasbourg (3), (9), (10), (46) on the polysaccharide is schematised on Fig.6.
Infectious Nucleic Acids of Escherichia coli Bacteriophages
Authors: Benzinger R. , Delius H. , Jaenisch R. , Hofschneider P. H.
Published: March 2005

Various strains of Escherichia coli were grown to a titer of 5 × 108 cells/ml in peptone media. Following osmotic shocks (peptone → water → 0.4 M sucrose) or lysozyme‐EDTA treatment in hypertonic media, the spheroplasts were stored at 4° and then tested for competence with ØX 174 and M 13 phage DNA and M 12 phage RNA. Lysozyme spheroplasts proved most effective and yielded the following maximum infectivities: for one Ø× 174 DNA molecule, 5 × 10−3 infectious DNA centers; for one M 13 DNA molecule, 10−5 infectious DNA centers; and for one M 12 phage RNA molecle, 2 × 10−5 infectious RNA centers. For many spheroplast preparations competence remained nearly constant for one week. One strain, E. coli 112–12 (λh)F+, retained a constant (though low) level of competence for months when frozen in 10% (v/v) dimethyl sulfoxide.

Ø× 174 DNA saturation experiments indicated that 0.05–1% of the lysozyme spheroplasts were competent. Between 12 and 20 minutes after the addition of a non‐saturating level of 32P‐labelled Ø× 174 DNA to such competent cell populations at 37°, 10–20% of the label was adsorbed. Noncompetent cell populations adsorbed no more than 4% of the added label. Prolonging the DNA adsorption time did not give a higher efficiency of DNA infection, because the cells began losing competence through a temperature‐dependent process.

More than 90% of the infectious Ø× 174 DNA centers reamined sensitive to DNAase during the first 20 minutes in DNA adsorption medium. Plating Ø× 174 DNA and M 12 RNA‐infected cells on eosin methylene blue agar during this period prevented the formation of phage, whereas the plaque‐forming ability of the mature phage was not inhibited. These observations may suggest an unusual permeability of the competent cells.

Inhibitors of phage RNA infection were removed by washing the competent cells. DNAase pretreatment of the spheroplasts increased their competence for infectious M 12 phage RNA.
The Importance of the Two Halves of Human Immunoglobin Light Chain in Recombination with the Heavy Chain
Authors: Ruffilli A. , Givol D.
Published: March 2005

The noncovalent recombination between heavy and light polypeptide chains of human immunoglobulin G was studied using a Bence‐Jones protein and a fragment representing its N‐terminal half. Under the conditions employed there is about 33% recombination of heavy chain with the Bence‐Jones protein to form 7 S molecules, and no recombination with the fragment. The results are discussed in view of the variability of light chains and its relation to the antibody site.
α‐Chain and Globin: Intermediates in the Synthesis of Rabbit Hemoglobin
Authors: Baglioni C. , Campana T.
Published: March 2005

The synthesis of hemoglobin by rabbit reticulocytes has been studied by means of pulse‐labelling and chase experiments. The reticulocyte lysate was fractionated into a top layer and a ribosomal fraction. The top layer was analysed by gel filtration on columns of Sephadex G‐75. At least two intermediates which were formed after short incubations and were chased into hemoglobin were separated by gel filtration. These intermediates have been identified with α chain and globin.

α Chain and globin have also been separated by chromatography on carboxymethyl‐Sephadex. This method of analysis has been used to measure the rate of appearance of these intermediates in the top layer fraction and their rate of removal in chase experiments. The presence of complete α chains on polyribosomes has been confirmed. The rate of appearance of α chains on polyribosomes and their rate of removal in chase experiments have been determined. These rates appear to be identical, suggesting that the same biosynthetic process is responsible for these rates.

The nature of the complete α chains present on polyribosomes has been investigated. These chains can be separated from peptidyl‐tRNA chains by gel filtration in a sodium dodecyl sulphate‐acetate buffer. This finding suggests that the complete chains are not bound to tRNA.

The experimental results support the following scheme of assembly of the hemoglobin molecule: α chains are released from polyribosomes on completion. These chains combine with β chains which are still being synthesized on polyribosomes and for this reason complete α chains are found on polyribosomes. αβ subunits are released from polyribosomes once the synthesis of β chains is completed. The subunits then combine with heme to form hemoglobin molecules. The combination with heme may be stepwise and, in this case, intermediates containing less than one heme every peptide chain are formed. Some evidence for the existence of heme‐containing intermediates has been obtained.

The significance of the combination of α chains with β chains at the polyribosome level is discussed. This phenomenon may have some regulatory function which may be of general importance in view of the similar finding that immunoglobulin light chains combine with heavy chains at the polyribosome level and that heavy chains are apparently not released independently from polyribosomes.
Conversion of Cholesterol to Bile Acids in Rat: Purification and Properties of a Δ4‐3‐Ketosteroid 5β‐Reductase and a 3α‐Hydroxysteroid Dehydrogenase
Authors: Berséus O.
Published: March 2005

A Δ4‐3‐ketosteroid 5β‐reductase active on 7α‐hydroxycholest‐4‐en‐3‐one and 7α,12α‐di‐hydroxycholest‐4‐en‐3‐one and a 3α‐hydroxysteroid dehydrogenase active on 7α‐hydroxy‐5β‐cholestan‐3‐one and 7α,12α‐dihydroxy‐5β‐cholestan‐3‐one have been isolated from the soluble fraction of a rat liver homogenate and have been partially purified by chromatography on TEAE‐cellulose and Sephadex G‐75.

The Δ4‐3‐ketosteroid 5β‐reductase required NADPH as cofactor and was inhibited reversibly by p‐chloromercuribenzoate. The enzyme preparation catalyzed the reduction of the double bond in a number of Δ4‐3‐ketosteroids of the C19, C21, C24, and C27 series. The reduction of 7α,12α‐dihydroxycholest‐4‐en‐3‐one in the presence of the Δ4‐3‐ketosteroid 5β‐reductase preparation was not inhibited by addition of 7α‐hydroxycholest‐4‐en‐3‐one and the reduction of 7α‐hydroxycholest‐4‐en‐3‐one was not inhibited by 7α,12α‐dihydroxycholest‐4‐en‐3‐one indicating that these substrates were reduced by different reductases present in the Δ4‐3‐ketosteroid 5β‐reductase preparation.

The 3α‐hydroxysteroid dehydrogenase required reduced pyridine nucleotide, NADPH being preferred, and was inhibited reversibly by p‐chloromercuribenzoate. The enzyme preparation catalyzed the reduction of the 3‐keto group in a number of 3‐ketosteroids of the C19, C21, C24, and C27 series. The rate of reduction was faster with 3‐keto‐5β‐steroids than with the corresponding 3‐keto‐5α‐steroids.
Regulation and Function of Pyruvate Kinase and Malate Enzyme in Yeast*
Authors: Fernández Ma J. , Medrano L. , Ruiz‐Amil M. , Losada M.
Published: March 2005

The concentrations of pyruvate kinase and malate enzyme have been determined in the yeast Rhodotorula glutinis and shown to vary considerably according to the carbon compounds consumed by the cells. The results so obtained, together with the kinetic studies of the synthesis of the enzymes, seem to indicate that pyruvate kinase is nutritionally induced by glucose whereas malate enzyme is repressed by glucose and by acetate.

In R. glutinis, the function of pyruvate kinase is to form pyruvate from phosphoenolpyruvate during glycolysis and that of malate enzyme to provide pyruvate from malate. The reverse reactions probably do not play any physiological role.

R. glutinis cells growing in a medium containing glucose could at the same time utilize aspartate as source of carbon. Under such conditions, asparate could not supply phosphoenolpyruvate nor pyruvate for biosynthetic and energetic purposes, because both phosphoenolpyruvate carboxykinase and malate enzyme are repressed by glucose, but could, however, provide oxaloacetate for biosynthesis and at the same time spare the pyruvate glycolytically formed by inhibiting its carboxylation. Once glucose was used up by the cells and after a clear diauxia, the yeast continued growing solely on the remaining aspartate. The disappearance of glucose from the medium determined a change in enzyme levels, pyruvate kinase being no longer induced and phosphoenolpyruvate carboxykinase and malate enzyme being no longer repressed. This enzymatic change allowed aspartate to fulfill by itself the functions of supplying energy and providing precursors for the synthesis of all cell constituents.
Enzymatic Synthesis of DNA with 4‐Thio‐Thymidine Triphosphate as Substitute for dTTP
Authors: Lezius A. G. , Scheit K. H.
Published: March 2005

When 4‐thio‐thymidine‐5′‐triphosphate is substituted for dTTP in the enzymatic synthesis of poly d(A‐T) by DNA polymerase, formation of a polymer is observed either by incorporation of radioactive labelled deoxyadenosine nucleotide or by a hypochromic effect at 260 mμ and at 335 mμ, the latter corresponding to the ultraviolet absorption of 4‐thio‐thymidine. Both the initial velocity and the extent of polymer synthesis are strictly dependent on the concentration of primer poly d(A‐T). The product for which the designation poly d(A‐4thioT) is proposed contains deoxyadenosine‐ and 4‐thio‐thymidine nucleotide in equal amounts and bears a close resemblance to the parent d(A‐T) polymer with respect to temperature induced helix coil transition. Evidence for enzymatic formation of a hybrid consisting of poly d(A‐BrU) and poly d(A‐4thioT) is given by melting profiles. Poly d(A‐4thioT) is a poor primer for synthesis of poly d(A‐T) and, if dTTP is replaced by 4‐thio‐thymidine triphosphate incorporation of labelled deoxyadenosine nucleotide becomes almost negligible. Moreover, 4‐thio‐thymidine triphosphate cannot replace dTTP when calf thymus DNA or the homopolymer double strands are used as templates. These results cannot be interpreted merely in terms of the classical rules of base pairing, but additional restrictions, e. g. influence of nearest neighbors, might be operating in the enzymatic reaction.
Studies on the Relationship betweeen Activity and Structure of Liver Acetyl‐CoA Carboxylase
Authors: Numa S. , Goto T. , Ringelmann E. , Riedel B.
Published: March 2005

Alterations in the state of aggregation of liver chicken acetyl‐CoA carboxylase in relation to its catalytic activity were investigated in the presences of various components participating in the carboxylation reaction. In the absence of citrate, ATP plus Mg2+, which inhibits the carboxylase, promotes dissociation of the enzyme. The inhibition as well as the dissociation by ATP plus Mg2+ is prevented by simultaneous addition of either acetyl‐CoA or ADP. The effect of ADP is not observed in the presence of a large amount of bicarbonate.
Structural Protein of Wild‐Type (ρ+) and Respiration‐Deficient (ρ−) Yeast Mitochondria
Authors: Tuppy H. , Swetly P. , Wolff I.
Published: March 2005

Structural protein prepared from mitochondria of wild‐type baker's yeast contains several components which are separable by electrophoresis on polyacrylamide gels. One of these components is absent from structural protein preparations obtained from a cytoplasmic “petite” mutant.

Rabbit antisera against the structural protein of yeast mitochondria are capable of agglutinating yeast mitochondria and human erythrocytes coated with yeast mitochondrial structural protein. The agglutination of coated erythrocytes can be inhibited by absorption of antisera with structural protein. With the help of this sensitive haemagglutination‐inhibition technique the antigenic properties of structural protein from mitochondria of the wild‐type yeast and the “petite” mutant were compared. It was found that at least one of the antigenic determinants present in the mitochondrial structural protein preparations from wild‐type yeast is lacking in those obtained from the mutant.
Purification of the Gramicidin S Synthesizing Activity in Bacillus brevis Extracts
Authors: Bredesen J. E. , Berg T. L. , Figenschou K. J. , Frøholm L. O. , Laland S. G.
Published: March 2005

A 20 fold purification of the gramicidin S synthesizing activity in a 11,000 ×g supernatant from Bacillus brevis has been achieved. The purification procedure involves precipitation of undesired material with streptomycin sulphate, followed by precipitation of gramicidin S synthesizing activity at 40% (NH4)2SO4 saturation and fractionation on DEAE‐Sephadex. All gramicidin S synthesizing activity appeared in one peak. It incorporated labelled phenylalanine, proline, valine, ornithine and leucine to the same extent into gramicidin S. The gramicidin S synthesizing activity was found to be stable in a 0.05 M potassium phosphate buffer (pH 7.1) containing glutathione (0.01 M) and glycerol (20%, v/v) for at least 3 days at + 4° and four weeks at −20°.
Kinetic Studies of Pigeon Liver NAD Kinase
Authors: Apps D. K.
Published: March 2005

A procedure for the purification of NAD kinase from pigeon liver acetone powder is described. Although not homogeneous, the preparation is largely free of enzymes which interfere with the assay of NAD kinase. The kinetics with magnesium as the activating ion are described: from Interrelationships between the kinetic coefficients, the modes of inhibition by NADH, NADPH and ADP, and the independent variation of Vmax and two Kms with pH, it is inferred that NAD kinase acts by a random addition, rapid equilibrium mechanism.
Antibodies to Enzymatic Breakdown Products of Yeast tRNA
Authors: Hernandez R. , Bürgin‐wolff A. , Just M.
Published: March 2005

Rabbits immunized with yeast tRNA without a protein carrier produced complement fixing antibodies which did not react with intact tRNA but with its oligonucleotides formed after cleavage with pancreas ribonuclease. The enzymatic breakdown products were fractionated according to chain length in 7 M urea. Penta‐ or hexanucleotides turned out to be the smallest molecules being able to fix complement with anti‐tRNA antibodies. An upper limit for reactivity with antibody is presumably constituted not by size but by tertiary configuration of the tRNA molecule.
Cholinesterase of Squid Optical Ganglia
Authors: Turpaev T. M. , Abashkina L. I. , Brestkin A. P. , Brick I. L. , Grigorjeva G. M. , Pevzner D. L. , Rozengart V. J. , Rozengart E. V. , Sakharov D. A.
Published: March 2005

Optical ganglia of squid (Ommatostrephes sloanei‐pacificus) possess very high cholinesterase activity.

Squid cholinesterase is located on the level of layers r2–r3 of plexiform zone in ganglion cortex.

The activity of catalytic site of squid cholinesterase was equal to 1.2 × 105 min−1.

The hydrolysis rates of acetylcholine, butyrylcholine and acetyl‐β‐methylcholine in the presence of squid cholinestarse were the same (in optimal substrate concentrations). But their Km values were different (the lowest ones for acetylcholine); squid cholinesterase did not hydrolyze benzoylcholine.

All organophosphorus inhibitors studied possessed a high activity with regard to squid cholinesterase. It was shown that O‐ethyl‐S(3,3‐dimethyl‐butyl)‐methyl‐thiophosphonate (LG‐56) is a selective inhibitor of squid cholinesterase.

Substrate and inhibitor specificity and catalytic activity of squid cholinesterase differed essentially from those of acetylcholinesterase and serum cholinesterase.
Altération par mutation de la régulation de la biosynthèse des cytochromes par les cultures anaérobies d'une bactérie anaérobie facultative
Authors: Chippaux M. , Pichinoty F.
Published: March 2005

Starting with a wild strain of Hafnia having the phenotype: nitrate reductase A +, nitrate reductase B +, chlorate reductase C +, tetrathionate reductase +, fumarate reductase +, hydrogen lyase +, we have isolated two kinds of mutants having the following phenotypes: (1) A –, B +, C +, tetrathionate reductase +, fumarate reductase +, hydrogen lyase +; (2) A –, B –, C –, tetrathionate reductase –, fumarate reductase +, hydrogen lyase –. The wild strain and the mutants of category 1 can use nitrate, nitrite, fumarate, and tetrathionate as hydrogen acceptors under anaerobic conditions. The mutants of category 2 can also reduce nitrite and fumarate, but cannot use nitrate and tetrathionate as hydrogen acceptors.

We have studied the effects of nitrate, nitrite, fumarate, and tetrathionate on the biosynthesis of cytochromes a2, b1, b‐562, and c‐550 using anaerobic cultures of the wild strain and several mutants of the above two categories. The following observations were made: (a) Wild strain. Neither tetrathionate nor fumarate have any effect. Nitrate, on the other hand, markedly reduces the levels of cytochromes a2, b‐562, and c‐550. As for nitrite, it causes a decrease in levels of cytochromes b‐562 and b1, but a strong increase in that of cytochrome c‐550. It is as though nitrate abolishes the effects of nitrite on the biosynthesis of cytochromes b1 and c‐550. (b) Mutants of category 1. Nitrate causes the cytochrome a2 band to disappear, but does not influence the biosynthesis of cytochromes b‐562 and c‐550. Apart from the effect of nitrate on the formation of cytochrome a2, the different hydrogen acceptors caused no detectable spectral changes. (c) Mutants of category 2. We obtain essentially the same spectra as with the wild strain except when the cultures are nitrite‐grown. In this case, nitrite has the same effect as nitrate because it represses the biosynthesis of cytochrome c‐550.

It is clear from this work that the two mutant phenotypes studied have profound and different effects in their regulation of the biosynthesis of cytochromes.
Intramembrane pH Changes and Bromthymol Blue Translocation in Liver Mitochondria
Authors: Azzone G. F. , Piemonte G. , Massari S.
Published: March 2005

The effect of pH and of ionic strength on the degree of binding of bromthymol blue to the mitochondria has been studied. Changes of the functional state of the mitochondria affect the degree of binding of bromthymol blue.

The changes of light absorbance of bromthymol blue have been followed under conditions where translocation of ions other than protons where minimized and in media with high buffering power. The transition from the de‐energized to the energized state, due to the addition of succinate or ATP to rotenone treated mitochondria, results in a decrease of absorbance of the alkaline form of the dye whereas the transition from the energized to the de‐energized state, due to the addition of respiratory chain inhibitors or of uncouplers is accompanied by an increase of absorbance. Several parameters of the phenomenon have been investigated including the competition between uncouplers and substrate anions. The direction of the change of absorbance is inverted when the ionic strength or the buffer strength of the medium are decreased and when the pH of the medium is increased.

The changes of absorbance may be expression of pK shifts of the dye, due either to membrane potential or to changes in the hydrophobic regions of the crystal membrane.