bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Histone Messenger RNA from HeLa Cells
Authors: Breindl Michael , GALLWTTZ Dieter 
Published: March 2005

Histone mRNA from HeLa cells synchronized by a double thymidine block has been identified and characterized. Its synthesis starts immediately after the cells are released from the thymidine block.Pulse labelling for 60 min with [5‐3H]uridine at different times of the S‐phase showed that radioactively labelled histone mRNA enters polyribosomes with a maximum during the first hour of S‐phase, i.e., before the maximum of DNA synthesis.Histone mRNA sediments on sucrose gradients in the 8–10‐S region and separates on polyacrylamide gels into three RNA species with molecular weights of about 1.5 × 105, 1.8 × 106, and 2.1 × 105, respectively.Polyribosomal 8–10‐S RNA from synchronized cells in S‐phase directs the synthesis of histones in a rabbit reticulocyte cell‐free system. Histones synthesized in vitro were identified by polyacrylamide gel electrophoresis and by high‐voltage paper electrophoresis of tryptic peptides. Polyribosomal 8–10‐S RNA from cells in which DNA synthesis was blocked by thymidine or hydroxyurea had only a marginal histone mRNA activity.
Studies on Soybean Trypsin Inhibitors
Authors: Koide Takehiko , IKENAKA Tokuji , Tsunasawa Susumu 
Published: March 2005

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion‐exchange chromatography on Dowex 50X2 or by gel filtration on Bio‐Gel P‐4. Further purification of the peptides was carried out by gel nitration on Sephadex G‐25 or by high‐voltage paper electrophoresis at pH 3.6 and 6.5.Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy‐peptidase digestion technique.Overlapping peptides necessary for the alignment of the tryptic peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio‐Gel P‐4, and their amino acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino‐terminal region of the inhibitor consisting of 84 amino acid residues. The reactive site (Arg63‐Ile64) of the inhibitor to trypsin was involved in this region.
Studies on Soybean Trypsin Inhibitors
Authors: Koide Takehiko , IKENAKA Tokuji 
Published: March 2005

For the elucidation of the amino‐acid sequence of the carboxyl‐terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion‐exchange chromatography on Dowex 50X2 or by gel nitration on Bio‐Gel P‐4.Further fractionation and purification of the peptides were performed by ion‐exchange chromatography on Dowex 1X2, by gel filtration on Bio‐Gel P‐2 or by high‐voltage paper electrophoresis at pH 1.9 and 3.6.Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino‐acid sequences were determined by the direct Edman method and by carboxy‐peptidase digestion technique.Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D.Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino‐acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino‐acid sequence of the carboxyl‐terminal region of the inhibitor.The complete amino‐acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman‐Birk inhibitor, another well‐known soybean proteinase inhibitor.
Metabolic Regulation of Steroidogenesis in Adrenocortical Carcinoma Cells of Rat
Authors: Sharma Rameshwar K. 
Published: March 2005

The effect of adrenocorticotropin and cyclic AMP on the transformation of pregnenolone to corticosterone has been studied in adrenocortical carcinoma and isolated normal adrenal cells of rat. This has been carried out using both chemical and double labeling techniques. Adrenocorticotropin or cyclic AMP did not shown any effect on the corticosteroidogenesis produced from pregnenolone in the isolated adrenal cells whereas the synthesis of deoxycorticosterone and corticosterone was inhibited by adrenocorticotropin in adrenal tumor cells. A similar inhibitory effect of adrenocorticotropin was observed when progesterone was used as a precursor for corticosteroidogenesis in tumor cells but the incorporation of deoxycorticosterone into corticosterone was not inhibited by this substance. These studies, therefore, suggest that the site of inhibition of adrenocorticotropin on corticosterone synthesis in the tumor lies between progesterone and deoxycorticosterone.Cyclic AMP also inhibited the synthesis of corticosterone from pregnenolone but no inhibition of deoxycorticosterone synthesis was observed. Similar results were obtained with N6,2′‐O‐dibutyryl adenosine 3′: 5′‐monophosphate. It is suggested that the mechanism of inhibition of Corticosteroidogenesis by adrenocorticotropin in the tumor may be different from that of cyclic AMP.
Kinetics of the Interaction between Pig‐Plasma Benzylamine Oxidase and Substrate
Authors: Lindström Anders , Olsson Bengt , Pettersson Gösta 
Published: March 2005

The rapid absorbance changes in the visible region observed on mixing pig plasma benzylamine oxidase with substrate under anaerobic conditions are due to reduction of a single chromophore, centered around 470 nm, in a single reaction phase reflecting a rate‐limiting combination of substrate to the enzyme. The second‐order rate constant of 1 s−1 mM−1 for this process is unaffected by the presence of oxygen.
Under aerobic conditions, addition of substrate to the enzyme leads to 470‐nm absorbance changes exhibiting a biphasic time‐dependence. A rapid phase of transient formation of the enzyme · substrate complex is followed by a slow readjustment of the steady‐state level of free enzyme, caused by substrate and oxygen consumption during the reaction.
The kinetic data obtained provide strong evidence that substrate‐binding to benzylamine oxidase is practically irreversible. They further indicate the presence of a single kinetically significant substrate‐ and oxygen‐independent rate‐limiting reaction step in the catalytic mechanism, with a velocity constant in the order of 1 s−1.
A valence change of enzyme‐bound copper could not be detected at any stage of the reaction between benzylamine oxidase and substrate, irrespective of whether or not oxygen was present in the reaction solutions.
Polyelectrolyte Behaviour of Phosvitin
Authors: Giancotti Vincenzo , Quadrifoglio Franco , Crescenzi Vittorio 
Published: March 2005

The results of spectroscopic and microcalorimetric measurements on dilute aqueous solutions of phosvitin are described. Our infrared and circular dichroism spectra show that the phosphoprotein chains may assume either random‐coiled or partially helical or β‐structures depending upon the pH. The transition to the low pH β‐form occurs with a distinct cooperative character.The strongly interdependent ionization of fixed charges densely spaced along phosvitin chains, typical of highly charged polyelectrolytes, is well illustrated by the calorimetric enthalpy of protonation data.The interaction of phosvitin with acridine orange in dilute aqueous solution (pH ∼5) has been studied by means of spectral and equilibrium dialysis experiments. For relatively low polymer to dye concentration ratios, bound acridine orange exhibits γ‐type spectrum thus suggesting aggregation of dye molecules onto the protein. Gradual decrease in the extent of bound acridine orange aggregation is produced by increasing the concentration of the protein; the monomerically bound form is hardly obtained, however, even at relatively high phosvitin concentrations. Binding curves reveal a cooperativity character for the dye‐protein interaction.It is suggested that about 60% only of phosphomonoester side groups of phosvitin are available for acridine orange binding, these being characterized by a high “stacking” coefficient.The results are tentatively discussed on the basis of present information on phosvitin chemical composition and conformation.
Magnetic‐Circular‐Dichroism Studies of Cytochrome c and Cytochrome b2
Authors: Risler Jean‐Loup , Groudinsky Olga 
Published: March 2005

In spite of the very different conformation of the polypeptide chain around the heme‐ligand chelate, the magnetic circular dichroism (MCD) spectra of native cytochrome c and of its heme undecapeptide dissolved in 3.3 MN‐acetyl‐dl‐methionine are identical in the Soret region. This indicates that the MCD spectrum of the heme group (in the Soret band) is not sensitive to changes in the protein conformation as long as the proper ligands are bound to the iron atom.On the contrary, the MCD spectrum of holocytochrome b2 differs from that of its derivatives lacking the FMN group; in the Soret region a MCD band at 365 nm, observed only with the holo‐enzyme, is assigned to the δ absorption band of the heme group rather than to a flavin transition. If, as shown with cytochrome c, the MCD spectra are independent of the protein conformation, the appearance of this band upon binding of FMN can be tentatively explained by a perturbation exerted on the heme group by the flavin itself. This would imply a close proximity of the two prosthetic groups.
Oxidation of Acetaldehyde by Rat‐Liver Mitochondria in Relation to Ethanol Oxidation and the Transport of Reducing Equivalents across the Mitochondrial Membrane
Authors: Grunnet Niels 
Published: March 2005

The primary product of the well‐known oxidation of acetaldehyde by rat liver mitochondria is acetate. About 50% of the acetate formed from acetaldehyde is converted to ketone bodies under the experimental conditions used.
The oxidation of acetaldehyde by rat liver mitochondria is catalyzed mainly by an aldehyde: NAD oxidoreductase present in the matrix compartment of the mitochondria. About 20% of the total mitochondrial acetaldehyde dehydrogenase activity may be confined to the outer membrane or the intermembrane space.
The enzyme has a broad substrate specificity, pH‐optimum at about pH 9.0 and is inhibited by tetraethylthiuram disulfide, chloral hydrate and arsenite. The enzyme is steroid‐insensitive.Two Km‐values for acetaldehyde, below 0.1 μM and 1.0 mM, and two Km‐values for NADM+, 2 μM and 50 μM, were obtained with a partially purified 100000 ×g supernatant. The presence of two aldehyde dehydrogenases in rat liver mitochondria could not be shown.
Oxidation of acetaldehyde by rat liver mitochondria causes an inhibition of the production of 14Co2, from [14C]palmitate. The inhibition can be accounted for by dilution of the acetyl‐coenzyme A pool of the mitochondria.The 3‐hydroxybutyrate/acetoacetate ratio during acetaldehyde oxidation is kept at the same level as during oxidation of oleate plus coenzyme A.
The mitochondrial acetaldehyde dehydrogenase activity is 80% of the total acetaldehyde dehydrogenase activity of rat liver. The results are discussed with regard to the oxidation of acetaldehyde and to the transfer of reducing equivalents from cytosol to mitochondria during ethanol metabolism.
Multiple Effects of Rifamycin Derivatives on Animal‐Cell Metabolism of Macromolecules
Authors: Busiello Enzo , Di Girolamo Adele , Di Girolamo Mario , Fischer‐Fantuzzi Lia , Vesco Cesare 
Published: March 2005

A group of rifamycin derivatives were tested against the nucleolar and nucleoplasmic RNA polymerases from three types of animal cells. It was found that several compounds effectively inhibited such enzymes without substantial discrimination. In addition, the same derivatives inhibited polyphenylalanine synthesis in an animal cell‐free system.Experiments with intact Hela cells showed that the synthesis of RNA, the processing of rRNA, the synthesis of DNA and the uptake of nucleosides were inhibited by rifamycins.It is concluded that the specificity of rifamycin effects obtained at the concentrations that appear to be necessary to interfere with normal (or virus‐directed) processes in animal cells can be seriously questioned.
Subunit Structure and Amino‐Acid Composition of Crystallized Human‐Muscle Glycogen Phosphorylase
Authors: Assaf Sáid A. , Yunis Adel A. 
Published: March 2005

The subunit structure and molecular weight of the a and b forms of human muscle glycogen phosphorylase were investigated.

Electrophoresis in the presence of sodium dodecylsulfate gave a molecular weight of 94 500 for the monomer. When studied at 1 mg/ml and 20 °C by high‐speed sedimentation equilibrium, sucrose density gradient ultracentrifugation and specific activity‐concentration dependence, both a and b forms of the human enzyme were found to exist as dimers.
A slight association of human phosphorylase a was observed in the high‐speed sedimentation equilibrium values of the z‐average molecular weight (262000 for Mmacr;zvs 188000 daltons for Mmacr;w). Some association of the a enzyme was also detected by schlieren sedimentation velocity when the enzyme was studied at 5 mg/ml.
Human phosphorylase b could be fully associated to tetramer at 20 °C when Pi, Mg2+ and AMP were added but not in the presence of NaF and AMP which were found to be effective in tetramerizing rabbit phosphorylase b.
Another important difference from rabbit phosphorylase was the significantly lower proline content of the enzyme from human muscle.
The Subunit Structure of the Escherichia coli K12 Pyruvate‐Dehydrogenase Complex
Authors: Vogel Otto , Henning Ulf 
Published: March 2005

It is shown that the dihydrolipoamide dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex consists of identical polypeptide chains (molecular weight: 5.6 × 104).Amino acid and tryptic peptide analyses gave a minimum chemical molecular weight agreeing with that based on FAD content of the enzyme or that obtained by dodecylsulfate polyacrylamide gel electrophoresis. Carboxypeptidase degradation revealed one C‐terminus ‐Lys‐Lys‐CO2H per 56000 molecular weight of protein.
Molecular Weight and Subunit Structure of the Malate‐Lactate Transhydrogenase
Authors: Allen S. H. George 
Published: March 2005

The molecular weight of the malate‐lactate transhydrogenase as determined by sedimentation‐diffusion and high‐speed‐equilibrium methods is approximately 70000. Dissociation of the protein with guanidine‐HCl, sodium dodecylsulfate or citraconic acid anhydride yields a homogeneous preparation for which molecular weight values ranging from 30000 to 43000 have been found. The transhydrogenase thus appears to be a dimer, composed of two subunits and an NAD+/NADH equivalent binding weight of 35000.Addition of a 33‐fold excess of citraconic acid anhydride based on the lysine content of the transhydrogenase was necessary for complete dissociation. This was accompanied by a complete loss of enzymatic activity and release of the bound NAD+/NADH, which, in the native enzyme, is very tightly bound. Attempts to restore enzymatic activity as well as to reform the dimer structure after removal of the citraconyl groups, even in the presence of NAD+ were unsuccessful. Partial citraconylation while destroying most of the enzymatic activity did not cause release of the NAD+/NADH prosthetic groups. The enzymatic activity of these preparations can be partially restored upon removal of the citraconyl groups. Apparently, removal of the prosthetic groups is associated with irreversible denaturation and the transhydrogenase represents a case where native configuration is extremely dependent on the binding of the prosthetic group.
Thioredoxin Reductase from Rat Liver
Authors: Larsson Agne 
Published: March 2005

The thioredoxin system was first identified in microorganisms where it participates in redox reactions, such as ribonucleotide reduction, transferring electrons from NADPH to the product formed. It contains two protein components, thioredoxin and thioredoxin reductase. A similar hydrogen transport system has now been demonstrated in rat liver. Using the liver thioredoxin previously isolated as a means of assaying thioredoxin reductase this enzyme has now been purified from the same source. A 100‐fold‐purification of thioredoxin reductase was obtained by treatment at pH 5 and chromatography on Sephadex and on DEAE‐cellulose. Thioredoxin reductase was found to be specific for NADPH.Ribonucleotide reductase, thioredoxin and thioredoxin reductase constitute a system necessary for the biosynthesis of deoxyribonucleotides. During liver regeneration, the level of ribonucleotide reductase was increased approximately 20‐fold. In contrast, the levels of thioredoxin and thioredoxin reductase remained constant.
Propriétés des formes allongées de l'acétylcholinestérase en solution
Authors: Bon Suzanne , Rieger François , Massoulié Jean 
Published: March 2005

We present here several molecular properties of the asymmetric acetylcholinesterase forms, which we described in our previous publications [1–3]. From molecular sieve experiments, we determined the Stokes radius and compared its value with that calculated from micrograph measurements [3] for the A form which is a tetramer associated to an elongated element, called the “tail” (structure en grappe). A good agreement is obtained with a prolate ellipsoid of identical volume and length; this indicates that the acetylcholinesterase is, in solution, a rigid structure.We also estimated the partial specific volume, v̄, of acetylcholinesterase, by two independent methods: first, by the difference of sedimentation velocity in gradients of different overall density (H2O, 2H2O); secondly by absolute density measurements in CsCl equilibrium gradients. Both methods are in reasonable agreement and yield a value of 0.74–0.75 for all forms of the enzyme. This enabled us to calculate, from sucrose gradient data, the sedimentation constant in water at 20 °C. Combining the values of the Stokes radius, sedimentation constant, and partial specific volume, we calculated the molecular mass of A, C, and D as 430000, 780000, and 1100000. These values justify our hypothesis that these forms possess one, two, and three tetramers, associated with the “tail”.The methods used in this work are all based on activity measurements, which is a major advantage since only minute quantities of protein are needed. However, these are mainly comparative determinations, and it is difficult to ascertain the precision of the published data for the reference proteins.
Oxidative Phosphorylation in Bacteria which Contain Different Cytochrome Oxidases
Authors: Meyer David J. , Jones Colin W. 
Published: March 2005

The oxidative phosphorylation efficiencies of seven species of bacteria, selected on the basis of their cytochrome oxidase composition, were determined from measurements of growth efficiencies with respect to oxygen (YO2) and →H+/O quotients of washed cell suspensions.
Organisms which contained either cytochrome oxidase aa3 (Bacillus subtilis, Microbacterium thermosphactum) or o (Xanthomonas hyacynthi, Acinetobacter lwoffi, Escherichia coli and Kurthia zopfii) as their sole terminal oxidase exhibited P/O ratios of approximately 3 whereas Acetobacter T71, which contained cytochrome oxidase a1, as its major oxidase, exhibited a P/O ratio of approximately 1.
The synthesis of cytochrome oxidases a1, and a2 by E. coli under oxygen‐limited conditions was accompanied by a sharp decrease in whole‐cell energy‐conversation efficiency. A similar decrease in efficiency was also observed as a result of the increased use by K. zopfii of a second oxidase, insensitive to cyanide and carbon monoxide, which has not hitherto been reported.
The possibility that the different cytochrome oxidases may terminate respiratory chains of different redox span or may be associated with separate phosphorylating or non‐phosphorylating chains is discussed in relation to the general properties and physiological functions of these oxidases.
Nucleoside‐Catabolizing Enzymes in Salmonella typhimurium Induction by Ribonucleosides
Authors: Nygaard Per 
Published: March 2005

Addition of cytidine, uridine or adenosine to glycerol‐grown cultures of Salmonella typhimurium induces the synthesis of cytidine deaminase and uridine phosphorylase (the cyt‐enzymes) as well as the synthesis of thymidine phosphorylase, deoxyriboaldolase, phosphodeoxyribomutase and purine nucleoside phosphorylase (the deo‐enzymes).The inducing effect of adenosine is more pronounced in mutant strains deficient in adenosine catabolism.A regulatory mutant which synthesizes the cyt‐enzymes constitutively has been isolated. This mutant also synthesizes the deo‐enzymes in elevated amounts. However, the level of the deo‐enzymes in the regulatory mutant like in wild‐type cells can be raised further if thymidine is added to the medium.The synthesis of the cyt‐enzymes appears to be sensitive to catabolite repression.From induction experiments carried out in mutant strains restricted in their metabolism of cytidine and uridine it appears that both compounds may act as inducer.Strains unable to metabolize cytidine or uridine contained increased levels of the cyt‐ and the deo‐enzymes even in the absence of external inducer. This is most likely due to endogenous induction by cytidine or uridine.
Biohydrogenation of Sterols by Eubacterium ATCC 21,408—Nova Species
Authors: Eyssen Hendrik J. , Parmentier Guido G. , Compernolle Frans C. , de Pauw Guido , Piessens‐Denef Maria 
Published: March 2005

Biohydrogenation of Δ5‐steroids was studied in vitro by incubating various steroids with a pure culture of Eubacterium 21,408 in a brain‐thioglycollate medium under anaerobic conditions.

Eubacterium 21,408, a strictly anaerobic bacterium, was isolated from rat cecal contents. It differed from any previously described Eubacterium species by its requirements for a Δ5‐3β‐hydroxy steroid.
Eubacterium 21,408 reduced the 5,6‐double bond of cholesterol, campesterol, β‐sitosterol and stigmasterol, exclusively yielding the corresponding 5β‐saturated derivatives. Similarly, the 4,5‐double bond of allocholesterol was reduced to yield coprostanol. Neither the 7,8‐double bond in 7‐dehydrocholesterol or in lathosterol, nor the 22,23‐double bond in the side chain of stigmasterol were reduced.
In the absence of a 3‐hydroxyl function (e.g. in cholest‐5‐ene), when the 3‐hydroxyl function was in the 3α‐position (e.g. in epicholesterol) or was substituted (e.g. in 3β‐chloro‐cholest‐5‐ene and in cholesteryl esters), no saturation of the 5,6‐double bond was observed.
When incubated with Eubacterium 21,408, the carbonyl group of 4‐cholesten‐3‐one, was reduced to the corresponding 3β‐hydroxyl group along with saturation of the 4,5‐double bond. The bacterium also reduced the 3‐oxo group of cholestanone and coprostanone to a 3β‐hydroxyl group.