bluuebunny/crossref_metadata_2025 huggingface dataset:
An X‐Ray Small Angle Study of the Bacteriophages fr and R17
Authors: Zipper Peter , Kratky Otto , Herrmann Richard , Hohn Thomas
Published: March 2005

X‐ray small angle scattering was performed on solutions of the bacteriophages fr and R17. The scattering curves of the phages are nearly identical. The radius of gyration was found to be 105.2 Å, the molecular weight as obtained from a high precision measurement is 3.62 × 106. The radial excess electron density distribution, derived from measurements in 0.02 M Tris‐Cl buffer pH 7.2 containing 0.15 M NaCl and 1 mM EDTA, indicates that the phages have a mean outer radius of 131.7 Å and consist of a shell and a less dense core. The core consists of RNA as is shown by measurements in sucrose solutions. It contains only about 80% of the RNA, the remaining 20% are considered to penetrate into the shell. Models calculated assuming the mean outer radius of the RNA to be 107.8 Å yield a mean inner radius of the protein shell of 104.9 Å. The amount of water associated with the phages is found to be about 0.9 g/g.
Immunochemical Studies on Hen's Egg‐White Lysozyme
Authors: Strosberg Arthur D. , Van Hoeck Béatrice , Kanarek Louis
Published: March 2005

By selective nitration of egg‐white lysozyme it is possible to obtain five lysozyme derivatives. Nitration of tyrosine 20 or tyrosine 23 slightly lowers the enzymatic activity and the thermal stability of the protein, modification of tyrosine 53 has a more drastic effect. Lysozyme nitrated on tyrosine 20 or tyrosine 23 loses one antigenic site. These two residues appear to belong to the same antigenic determinant, as lysozyme nitrated on these two tyrosines reacts with the same antibodies than the two former derivatives. Tyrosine 53 belongs to a second antigenic determinant of the enzyme, as it appears from the reaction between trinitrated lysozyme and the antilysozyme antibodies.
Some Aspects of the Process of DNA Synthesis in Mammalian Cells
Authors: Harrison Paul R.
Published: March 2005

The process of DNA synthesis has been investigated in Ehrlich ascites tumour cells in suspension either in vivo or in vitro, in continuous cell lines growing as monolayers (HeLa and L cells), and in primary explants of normal embryonic tissue growing as monolayers.

Newly‐synthesized DNA was detected by pulse‐labelling with tritiated thymidine and isolated by extraction with phenol/sodium dodecyl sulphate at pH 8.0. By chromatography on methylated albumin/kieselguhr, rapidly‐labelled DNA has been resolved into four components. One component, P1, was eluted with bulk DNA by about 0.65 M NaCl. In the case of DNA labelled during a very short pulse, a second component, P2, was eluted by about 0.8 M NaCl. Two further components, (β1 and β2) were eluted by 0.75 M and 6 M guanidine thiocyanate. The proportion of the latter components in total rapidly‐labelled DNA decreased only slightly as the time of incubation with label was increased from 7 to 60 min.

P2 and the β components were precipitated by 2 M potassium acetate plus 35% ethanol, whereas about 70% of P1 remained in solution. Furthermore, the proportion of single‐stranded regions in the rapidly‐labelled DNA (measured by binding to nitrocellulose) decreased with increasing time of incubation with label. However, at all times, rapidly‐labelled DNA resembled double‐stranded DNA by chromatography on hydroxyapatite.

There was no evidence for a different mechanism of DNA synthesis in the normal cells and the malignant cells or continuous cell lines. However, the timing of labelling of P2 varied between the cells.
Spectroscopic Studies on the Reduction and Reformation of the Disulfide Bonds of Papaya Lysozyme
Authors: Barel André O. , Dolmans Marcel , Léonis José
Published: March 2005

The study of the reduction and reformation of the four disulfide bonds cross‐linking the single polypeptide chain of papaya lysozyme (molecular weight 28000) has been undertaken. The four disulfide bonds were quantitatively reduced and carboxymethylated. Various physicochemical measurements, such as optical rotatory dispersion, circular dichroism and fluorescence showed that the polypeptide chain of the reduced inactive papaya lysozyme appears to behave as a random coil. The conformational changes can be characterized as a complete loss of helical structure and an extensive exposure of the aromatic chromophores. Partial reduction of the protein (with splitting of, most probably, a single disulfide bond) does not affect the enzymatic activity. Physico‐chemical studies indicated that the secondary and tertiary structure of this derivative are very similar with those of the native enzyme.

Reoxidation of the completely reduced enzyme, on exposure to air, produces a protein which could not be distinguished from the native molecule with respect to optical rotatory dispersion, fluorescence, solvent perturbation and enzymatic activity. The results of this study show that the primary structure of papaya lysozyme is sufficient to direct the refolding of the reduced protein in order to reform the native three‐dimensional conformation.
Sites of Biosynthesis of Outer and Inner Membrane Proteins of Neurospora crassa Mitochondria
Authors: Neupert Walter , Ludwig George D.
Published: March 2005

Outer and inner membranes of Neurospora crassa mitochondria were separated by the combined swelling, shrinking, sonication procedure. Membranes were characterized by electron microscopy and by marker enzyme activities. A red carotenoid pigment was found to be concentrated in the outer membrane. The inner mitochondrial membrane was resolved into about 20 protein bands on polyacrylamide gel electrophoresis, whereas the outer membrane shows essentially one single protein band. Only negligible incorporation of radioactive amino acids occurs into outer membrane when isolated mitochondria are synthesizing polypeptide chains. In agreement with this observation labeling of outer membrane protein is almost entirely blocked, when whole Neurospora cells are incubated with radioactive amino acids in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Finally, the essential electrophoretic protein band from outer membrane does not become labeled when mitochondria are incubated with radioactive amino acids either in vitro or in vivo in the presence of cycloheximide. It is concluded that the vast majority, if not all, of the outer membrane protein is synthesized by the cytoplasmic system and that polypeptide chains formed by the mitochondrial ribosomes are integrated into the inner mitochondrial membrane.
Structure primaire de la caséine β bovine Séquence partielle
Authors: Brignon Ghislaine , Ribadeau Dumas Bruno , Grosclaude François , Mercier Jean‐Claude
Published: March 2005

This paper is the fifth of a series devoted to the primary structure of the bovine β‐casein A2. The first two communications dealt with the isolation, analysis and positioning in the peptide chain of the tryptic and “CNBr” peptides [1,2]. The last two described the sequence of 65 residues from the carboxyl‐terminus and of 32 residues from the amino‐terminus [3,4].

We report here a partial sequence which includes 156 residues (out of the 209 residues of the β‐casein A2) exactly positioned in the peptide chain. Two segments including 53 residues (from position 36 to 68 and from 73 to 92) remain to be analyzed.

In addition to the tryptic and “CNBr” peptides originating from β‐casein A2, two peptides issued from a tryptic digest of β‐casein B were used as starting material.

The position of the substitution His→Gln which differenciates the β‐casein A2 from the β‐casein A3, already given in a preliminary communication [5], is indicated, as it is for the substitution Ser→Arg which represents one of the two differences occurring between the β‐casein A2 and the β‐casein B.

Four of the five phosphorus atoms of the β‐casein A2 have been previously located in the NH2‐terminal tryptic peptide [4]. The fifth has been located at position 35. All the phosphorus atoms of the β‐casein A2 occur as phosphoserine.

The complete structure of the β‐casein A2 will be given and discussed in the next and last paper.
Studies on the Bacteriophage MS2
Authors: de Wachter Rupert , Merregaert Jozef , Vandenberghe Antoon , Contreras Roland , Fiers Walter
Published: March 2005

The 5′‐terminal sequence of bacteriophage MS2 RNA was established up to position 125. The overlap with the ribosomal binding site for the A‐protein cistron indicates that the first initiating A‐U‐G codon starts at position 130. A characteristic secondary structure with a high amount of base‐pairing is proposed for this 5′‐terminal fragment. The existence of a symmetrical structure at the 3′‐end of the complementary minus strand can be deduced. It is suggested that the latter structure forms the basis of a specific three‐dimensional folding, which may serve as a recognition site for the viral RNA polymerase complex. Both structures bear some analogy to the cloverleaf model for tRNA, especially the one of the complementary strand. Not only does it end on… C‐C‐A, but the general outline of the secondary structure is also similar, except for the length of the loops and the presence of one extra loop.
Evidence that Protein is Bound to the Rapidaly Labelled RNA in Polyribosomes in vivo
Authors: Olsnes Sjur
Published: March 2005

When ribosomal subunits were liberted from polysomes by puromycin treatment the rapidly labelled RNA banded in a CsCI gradient with the same low buoyant density as when the polysomes were treated with EDTA.

A complex of cRNA and ribosomes was prepared in vitro and incubated with a cytoplasmic postribosmal fraction. The low buoyant density of the cRNA after such treatment indicated that protein had been adsorbed. However, after prior sedimentation through sucrose, the cRNA banded with a considerably higher buoyant density indicating that most of the adsorbed protein had been removed during the sedimentation in sucrose.

It is concluded that the protein bound to the rapidly labelled RNA in polysomes cannot represent ribosomal strctural protiens, adsorbed nuclear material or cytoplasmic protein adsorbed during the homogenization. Most likely protien is bound to the rapidly labelled RNA in polysomes in vivo
Heterogeneity and Polymorphism of Human‐Liver Alcohol Dehydrogenase
Authors: Schenker Toni M. , Teeple Leslie J. , von Wartburg Jean‐Pierre
Published: March 2005

Seven isoenzymes of human liver alcohol dehydrogenase have been ascertained by means of starch gel electrophoresis and chromatography on ion‐exchange celluloses. Six of them have been isolated and purified.

Catalytic properties of the isolated isoenzymes and the results of their reversible monomerization suggest that they are formed by the possible dimeric combinations of three subunits A, B and B′.

The same isoenzymes as observed in normal livers have been found in livers containing the atypical, variant enzyme described previously. The predominating isoenzyme isolated from atypical livers exhibits the catalytic features characteristic for the atypical enzyme in liver homogenates. One isoenzyme with normal kinetic properties, however, is also found in atypical livers.
Affinity Chromatography of DNA‐Binding Enzymes on Single‐Stranded DNA‐Agarose Columns
Authors: Schaller Heinz , Nüsslein Christiane , Bonhoeffer Friedrich J. , Kurz Christina , Nietzschmann Irmtraud
Published: March 2005

Agarose gels containing immobilized single‐stranded circular DNA from phage fd or denatured calf thymus DNA were investigated for their use in the affinity chromatography of DNA‐binding enzymes. The DNA content of gel fragments is stable under the conventional conditions of enzyme purification. Single‐stranded DNA‐agarose columns have a high capacity to bind DNA‐specific proteins. They were used to differentiate between similar enzymatic activities in DNA‐free extracts from Escherichia coli. Preparative purification is described for the following enzymes: E. coli DNA polymerase I, DNA polymerase II, RNA polymerase, exonuclease III and T4 polynucleotide kinase. Enzyme purification was as high as 200‐fold, recovery of enzymatic activity was 75–100%.
Equivolumetric Glycerol and Sucrose Gradients for the B‐XIV and B‐XV Zonal Rotors
Authors: van, der Zeijst Ben A. M. , Bult Hidde
Published: March 2005

In equivolumetric gradients particles move through a constant volume per unit of time, proportional to their sedimentation coefficient. A total of 324 different equivolumetric glycerol and sucrose gradients for the B‐XIV and B‐XV zonal rotors have been obtained by computer calculations for particle densities at intervals of 0.10 from 1.20 to 2.00 g/ml, each at 5°C, 10°C and 20°C and at 5%, 10% and 15% (w/w) top concentration. Plots of the gradients which can directly be used for the construction of the gradients with a commercially available profile‐reading gradient‐forming apparatus have been made and are available from the authors. The form of the gradients can also be reconstructed from the parameters for construction of the gradients in a constant‐volume gradient mixer which are also given. The usefulness of the calculations is demonstrated by the results of separations of ribosomal subunits and of polysomes from the yeast Saccharomyces carlsbergensis.
Formes natives et globulaires de l'acétylcholinestérase dans la moëlle épinière et le cerveau de gymnote, Electrophorus electricus
Authors: Rieger François , Tsuji Shigeru , Massoulié Jean
Published: March 2005

Acetylcholinesterase was studied in subcellular fractions of the spinal cord and brain from electric eel: the enzyme species we found were identical to the native forms which were characterized in electric organ extracts. On the contrary, the soluble protein fraction contains only globular species: mainly Gb (11.8 S) and a variable proportion of G'b (7.7 S), depending on the age of the homogenate and the tissue studied. A significant proportion of Gb is also found in particulate fractions.

It seems probable that endogenous proteases cause the solubilization of the enzyme, yielding globular species Gb and G'b. Addition of benzethonium, an inhibitor of proteolytic enzymes, to spinal cord preparations results in a decrease of the proportion of globular species. This inhibitor also slows down the conversion of added “native” species A, C, and D into globular forms.

Using a modified Koelle staining technique, we have not been able to detect any soluble acetylcholinesterase activity in the cytoplasm of the spinal cord neurons in electron microscopy. Most of the activity seems to be bound to the endoplasmic reticulum, which further suggests that proteases are responsible for the formation of globular species in the spinal cord homogenate.
Consequences of Lysine Oversynthesis in Pseudomonas Mutants Insensitive to Feedback Inhibition
Authors: Hermann Monique , Thevenet Nicole J. , Coudert‐Maratier Michèle M. , Vandecasteele Jean‐Paul
Published: March 2005

Regulation of lysine biosynthesis was studied in Pseudomonas putida and Pseudomonas acidovorans. In both species, aspartokinase was subject to feedback inhibition by lysine and threonine and dihydrodipicolinate synthetase was inhibited by lysine.

P. acidovorans could not degrade lysine whereas P. putida degraded lysine through an inducible lysine oxygenase.

P. putida mutants with an aspartokinase insensitive to lysine inhibition were obtained. They did not excrete lysine but their lysine oxygenase was endogenously induced. However mutants obtained from a mutant lacking lysine oxygenase still did not excrete lysine.

P. acidovorans mutants with a dihydrodipicolinate synthetase desensitized towards lysine inhibition were obtained. From them double mutants with both dihydrodipicolinate synthetase and aspartokinase desensitized were also obtained. All of these mutants excreted lysine.
Purification of Methionyl‐tRNA Synthetase from Escherichia coli K12 by Affinity Chromatography
Authors: Robert‐Gero Malka , Waller Jean‐Pierre
Published: March 2005

An affinity column for the purification of methionyl‐tRNA synthetase has been prepared. Cyanogen‐bromide‐activated Sepharose 4B was reacted successively with hexamethylene‐diamine and N‐nitrophenylsulphenylmethionine succinimide ester. The nitrophenylsulphenyl protecting group of the polymer was removed under mild conditions with aqueous sodium thiosulfate. The affinity columns were equilibrated in 200 mM potassium phosphate buffer; this ionic strength proved to be sufficiently high to avoid the retention of methionyl‐tRNA synthetase by ion exchange.

On columns with high methionine concentration (8–20 μmol/ml gel bed), the enzyme was strongly retained but could be selectively eluted by addition of 20 mM methionine to the buffer. However this resulted in excessive dilution of the enzyme. Highly purified enzyme could be obtained in high yield by lowering the methionine substitution of the polymer to 4.5 μmol/ml gel and by introducing the affinity chromatography after the first DEAE‐Sephadex step of the conventional purification procedure. Under these conditions no retention but sufficient retardation of the enzyme on the column was observed.

The possibility of generalizing the method for the purification of other proteins recognizing an amino acid with its free amino group is discussed.
Sequence of Amino Acids of the NH2‐Terminal Region of a Mouse‐Clonal Immunoglobulin Heavy Chain
Authors: Bourgois Alain , Fougereua Michel , DE Preval Claude
Published: March 2005

The amino acid sequence of the cyanogen bromide fragments H1‐H3 from the γ chain of the plasmacytoma immunoglobulin MOPC 173 is reported. It represents the first 104 amino acid residues of this heavy chain, thus covering the major part of the VH region. This sequence allowed definition of a third subgroup of variability for the heavy chain pool, in common between mouse and human immunoglobulins.
Ribonucleoprotein Particles in HeLa Cells
Authors: Wengler Gerd , Wengler Gisela , Scherrer Klaus
Published: March 2005

We have investigated some properties of a class of cytoplasmic ribonucleoprotein particles in HeLa cells. After 3.5 h of labelling in the presence of 0.04 μg/ml actinomycin D to inhibit rRNA synthesis, about 20% of the newly synthesized cytoplasmic RNA, that sediments faster than 7 S, is contained in two types of particles sedimenting at 45 S and 30 S in sucrose gradients. We have examined this material in more detail with the following results.

The fixed particles band as two sharp peaks of density o= 1.440 g/cm3 for the 45‐S and density o= 1.460 g/cm3 for the 30‐S material. We call them the 45‐S‐M and 30‐S‐M particles, respectively.

The RNA of the 45‐S‐M and 30‐S‐M particles sediment in sucrose gradients as sharp peaks at 16 S and 12 S, respectively.

The synthesis of these two RNA species is inhibited by ethidium bromide, but resistant to low concentrations of actinomycin D. Two RNA species with these drug sensitivities and sedimentation characteristics have been found in the mitochondria of HeLa cells [1].

If cell homogenization and fractionation are carried out without detergents, the 45‐S‐M and 30‐S‐M particles are quantitatively bound to mitochondria.

Under most of the conditions commonly used for cell lysis and fractionation, these particles are extracted into the postmitochondrial‐cytoplasmic fraction.
Gene Linkage and Gene‐Enzyme Relations in the Fatty‐Acid‐Synthetase System of Saccharomyces cerevisiae
Authors: Kühn Lothar , Castorph Helga , Schweizer Eckhart
Published: March 2005

In Saccharomyces cerevisiae, the genetic linkage as well as the biochemical characteristics of nine different fatty‐acid‐synthetase complementation groups have been studied. By spore tetrad analysis, the alleles of these nine groups were established to be located on three genetically unlinked DNA regions on the yeast genome. These regions consist of one separate gene and two clusters comprising two and three adjacent cistrons, respectively. In both clusters the alleles are closely linked within less than one map unit. The cistrons of the first cluster can be assigned to structural genes of the dehydratase and the second reductase, those of the second cluster to the first reductase and two proteins involved in the condensation reaction. Mutants of the separate fas‐cistron produce a fully active fatty acid synthetase complex with all the component enzyme activities unimpaired under the assay conditions employed. The function of the gene product of this cistron is unknown, so far. From the pleiotropic fas‐mutants, a fatty‐acid‐synthetase complex with residual component enzyme activities could not be isolated. It is concluded what due to the polar transcription of the clustered cistrons in these mutants either none or only an incomplete aggregate is formed with the concomitant inactivation of residual component enzymes.
Steroid Metabolism in Rats Given [1‐2H2E]Ethanol
Authors: Cronholm Tomas , Makino Isao , Sjövall Jan
Published: March 2005

The incorporation of deuterium into bile acids and cholesterol in rats with a bile fistula given [1‐2H2]ethanol was studied by gas chromatography‐mass spectrometry. Deuterium‐labelling of cholic and chenodeoxycholic acids was observed with 0.03 g [1‐2H2]ethanol per kg body weight. The 5β position in both bile acids reached a deuterium excess of about 7 atoms per cent with 1 g [1‐2H2]ethanol per kg body weight. With this ethanol dose, the deuterium excess in the 3β position was 15 atoms per cent in chenodeoxycholic acid, and 9 atoms per cent in cholic acid. The deuterium excess in the 3β position of metabolites of 3‐keto‐5β‐cholanoic acid was about 13 atoms per cent. In the endogenous bile acids, several positions other than 3β and 5β were labelled, and the number of di‐ and trideuterated molecules relative to that of monodeuterated ones increased during the first 2 h after ethanol injection. Cholesterol in bile was also labelled in several positions, and the proportion of labelled molecules increased slowly to reach 8–14% 2–3 h after the injection of 1 g [1‐2H2]ethanol per kg body weight. The deuterium‐labelling of bile acids in positions other than 3β and 5β was greater than that of cholesterol. Fasting for 48 h, and fasting followed by glucose feeding had little influence on the deuterium incorporation into bile acids, where as pyrazole prevented it almost completely.

The results indicate that deuterium is transferred from ethanol via NADH to the NADPH that is utilized in the biosynthesis of cholesterol and bile acids. The higher and more variable labelling of the 3β than the 5β position may be explained by isotope effects or by the ability of the 3α‐hydroxysteroid dehydrogenase, involved in the reduction of the intermediary 3‐keto steroids, to utilize NADH. The difference between biliary cholesterol and bile acids with respect to labelling in positions other than 3β and 5β indicates that these compounds do not originate from the same cholesterol pool.

All metabolites of 3‐keto‐5β‐cholanoic acid carried a 3α‐hydroxy group. This indicates that the 3β‐hydroxy‐5β‐steroid dehydrogenase isoenzyme of alcohol dehydrogenase has no function in the reductive metabolism of bile acids. Furthermore, the increased production of NADH during ethanol metabolism did not induce a formation of 3β‐hydroxycholanoic acids.
Tyrosyl‐tRNA Synthetase of Escherichia coli
Authors: Chousterman Suzanne , Chapeville François
Published: March 2005

Using purified Escherichia coli tyrosyl‐tRNA synthetase it was found that the enzyme is a dimer containing apparently identical subunits of molecular weight 47000.

The study of the complexes formed between the enzyme and its substrates leads to the conclusion that there are two binding sites for tyrosyladenylate as well as for tRNATyr per molecule of enzyme. No binding of tyrosine alone could be observed, but a complex with ATP alone was characterized.
Reduced Bohr Effect in NO‐Ligated Chironomus Haemoglobin
Authors: Trittelvitz Eberhard , Sick Hinrich , Gersonde Klaus , Rüuterjans Heinrich
Published: March 2005

The low‐temperature electron‐spin‐resonance spectra of the NO‐derivatives of the monomeric Chironomus haemoglobins I, III, and IV being nearly identical to each other, indicate rhombic symmetry of the innermolecular electric field. At gz= 2.003 nine hyperfine lines (ΔH= 7 G) of nearly identical intensity are observed in the presence of 14NO, while 15NO produces two hyperfine lines (ΔH= 30 G) both showing a further splitting into three superhyperfine lines (ΔH= 7 G). These hyperfine structures represent the interaction of the unpaired electron with the N‐nuclei of the 5th and 6th ligand.

No changes of the electron‐spin‐resonance spectra were found in the range of pH 5 to 10, although haemoglobin III and IV are known to possess an alkaline Bohr effect. Thus in case of NO‐binding the Bohr effect is apparently reduced. With the aid of proton‐magnetic‐resonance studies of the Bohr proton binding site, His‐G2, in NO‐haemoglobin III it was demonstrated that the pK value of this group is shifted by only 0.15 units from that of deoxy‐haemoglobin. This shift is considerably lower as compared with that induced by CO‐ligation.
Freezing of Rat‐Liver Lysosomes to —196°C in the Presence and Absence of Dimethylsulphoxide
Authors: Osborne Jacqueline A. , Morris George J. , Lee Donald
Published: March 2005

The latency of acid phosphatase and β‐glucuronidase in homogenates of rat liver, prepared in 0.25 M glycerol‐2‐phosphate (pH 7.0), decreased after freezing to –196 °C. The latencies obtained were virtually independent of cooling rate in the range of 2–225 °C/min and the latency of acid phosphatase always suffered a greater decrease than that of β‐glucuronidase.

The fall in latency was always less in the presence of 7.5% (1.06 M) dimethylsulphoxide and and the protection afforded to the latency of acid phosphatase was greater than that given to β‐glucuronidase. The sedimentability of both enzymes responded to freezing in a manner similar to that found for latency.

The sedimentability of acid phosphatase decreased after exposure to high concentrations of sucrose or glycerol‐2‐phosphate. We suggest that this response, as well as the decrease in latency found after freezing and thawing, results from osmotic pressure gradients which arise when normally non‐penetrating molecules enter the lysosomes under a high concentration gradient.

The decrease in sedimentability of acid phosphatase after exposure to high concentrations of glycerol‐2‐phosphate was accompanied by a decrease in the specific total activity of the enzyme and it appears that this enzyme is readily inactivated in the presence of high concentrations of this substrate.
Inhibition of Acetylcholinesterase Activity by Aromatic Chelating Agents
Authors: Wermuth Bendicht , Brodbeck Urs
Published: March 2005

Form B of acetylcholinesterase from toluene‐treated electric organs of the electric eel was inhibited in a non‐competitive manner by chelating agents such as 1,10‐phenanthroline, 8‐hydroxyquinoline, 2,2′‐dipyridyl and salicylic aldehyde. The apparent inhibition constants ranged between 1.3 mM and 4.8 mM. Secondary plots revealed hyperbolic inhibition for 1,10‐phenanthroline, slope‐parabolic‐intercept‐linear inhibition for 8‐hydroxyquinoline, slope‐linear‐intercept‐hyperbolic inhibition for 2,2′‐dipyridyl and a linear inhibition pattern for salicylic aldehyde.

The direct participation of metal cations in the catalytic process could be ruled out on the following basis: the inhibition of the enzyme by chelating agents was rapid and reversible; it did not show any time‐dependent irreversible inactivation. Excess metal did not reverse the inhibition: metal · chelate complexes inhibited the enzyme similarly or even stronger as did the complexing agents by themselves. No inhibition, even at concentrations up to 0.1 M was observed with EDTA.

With 8‐hydroxyquinoline at pH 6.0 non‐linear double‐reciprocal plots were observed. The Hill coefficients were 1.01, 1.20, 1.30 and 1.32 for 0, 0.75, 1.50 and 3.00 mM 8‐hydroxyquinoline.

The inhibitory effects of these compounds were explained in terms of hydrophobic adsorption of aromatic nuclei at the active center and in terms of structural similarities to the substrate. The type of inhibition observed by these compounds is indicative of co‐operative binding of these ligands to acetylcholinesterase.
The Functions of rho in T7‐DNA Transcription in vitro
Authors: Darlix Jean‐Luc
Published: March 2005

The process of T7 DNA Transcription by Escherichia coli RNA polymerase in the presence of rho has been studied by means of hybridization and gel electrophoresis of the RNA synthesized. Transcription of T7 DNA is totally asymmetrical and restricted to the early region of the genome. The RNA produced is composed of five different species which are 600, 1100, 1500, 3000 and 4000 nucleotides long, and each species is homogeneous and initiated with ATP. The RNA species which are 1500, 3000 and 4000 nucleotides long are probably initiated at the same promoter whereas the species which are 600 and 1100 nucleotides long are initiated at two other distinct promoters. The RNA sequences with 600, 1100 and 4000 nucleotides are unique. The comparison of the RNA species made in vitro with those made in vivo upon infection of E. coli by T7 phage in the presence of chloramphenicol indicates that (a) the sequences of the RNA species with 1500 and 3000 nucleotides made in vivo are identical to those made in vitro, (b) the sequences of the RNA species with 600 and 1100 nucleotides synthesized in vivo are different from those made in vitro and (c) the sequences of the RNA species made in vivo are part of the RNA sequence which is 4000 nucleotides long and made in vitro. In view of the above results it is concluded that rho could be considered as a defence of the cell against phage infection.
Properties of the Calcium‐Independent ATPase of the Membranes of the Sarcoplasmic Reticulum Delipidated by the Nonionic Detergent Triton X‐100
Authors: Walter Horst , Hasselbach Wilhelm
Published: March 2005

When the sarcoplasmic reticulum membranes are solubilized in Triton X‐100 their ATPases are modified: the calcium‐dependent ATPase is activated whereas the basic ATPase is inhibited.

Phospholipids are removed from the solubilized sarcoplasmic membranes completely by chromatography on a sepharose column equilibrated with Triton X‐100. Thereby the activity of the calcium‐dependent ATPase is abolished and cannot be restored by addition of phospholipids.

The delipidated ATPase preparation, exhibits only low activity, which is not dependent on calcium but on magnesium alone. This calcium‐independent ATPase is activated to the level of the calcium‐dependent ATPase both by unsaturated and saturated fatty acids, by anionic phospholipids and various synthetic lipids.

The enhancement of the ATPase activity produced by lipids is related to the binding of these compounds to the membrane protein in amounts comparable with those of bound Triton as demonstrated by dialysis experiments with dodecylsulfate. When the concentration of Triton in the medium is lowered, the dodecylsulfate/protein binding ratio increases, while Triton is detached from the protein.

The activation of the calcium‐independent ATPase by saturated fatty acids is dependent upon the chain‐length of these aliphatic compounds. Only the long‐chain fatty acids are strong activators. Moreover, the hydrophilic group of lipids affects maximal activities. No activation is observed with lecithin or dodecanol, while alkyl‐sulfates or ‐phosphates activate most effectively.

The calcium‐independent ATPase differs from the calcium‐dependent ATPase in that it is insensitive to N‐ethylmaleimide, dinitrodithiobenzoic acid or salyrgan. The enzyme is strongly inhibited by prenylamine, sodium azide and silver ions. Similarly low alkali salt concentrations produce a strong reduction of the activity.

The relative rates, with which the nucleoside triphosphates are split by the calcium‐independent ATPase, differ considerably. As observed for the calcium‐activated ATPase. The dependence on the substrate concentration suggests a positive cooperativity concerning Mg · ATP (Hill coefficient 1.46). 50%, of maximal activity is obtained at 0.25 mM Mg · ATP. The enzyme is strongly inhibited by Mg · ADP.
The Mechanism of Action of Methionyl‐tRNA Synthetase
Authors: Lawrence Françoise , Blanquet Sylvain , Poiret Maryse , Robert‐Gero Malka , Waller Jean‐Pierre
Published: March 2005

The catalytic properties of native and trypsin‐modified methionyl‐tRNA synthetases were compared in the ATP‐PPi exchange reaction and in the reaction leading to aminoacyl‐tRNA formation performed under comparable experimental conditions.

In the ATP‐PPi exchange reaction, the two forms of the enzyme are virtually indistinguishable with respect to ionic requirements and Michaelis constants for ATP and methionine, while V values are in a ratio of 2 to 1 in favor of the native enzyme.

In the reaction leading to aminoacylation of tRNAfMet and tRNAmMet, the native and trypsin‐modified enzymes differ in several respects. In particular, a pronounced inhibition by ATP occurs with the native enzyme but not with the trypsin‐modified form. Also, the ratio of the V values measured under optimal conditions are closer to 4 than to 2, in favor of the native enzyme.

The ionic requirements for the aminoacylation reaction were examined in detail. Spermine, spermidine and NH4Cl, but not KCl, are able to sustain the formation of methionyl‐tRNA in the absence of magnesium, albeit at lower rates than in its presence. A pronounced synergistic effect of MgCl2 on the reaction catalyzed in the presence of spermidine or NH4Cl is described.
Relative Rates of Degradation of Mouse‐Liver Surface‐Membrane Proteins
Authors: Gurd James W. , Evans W. Howard
Published: March 2005

The relative rates of degradation of the proteins of liver membranes and soluble fractions were examined by using a double‐isotope labelling technique.

The proteins of plasma membrane and smooth microsomal fraction were more rapidly degraded than those of the mitochondrial and soluble fractions.

No great variation in the rates of degradation of the proteins of plasma membrane and microsomal subfractions was detected. The proteins of intercellular gap‐junctions (nexuses) were degraded slowly.

Extraction of plasma membranes with aqueous and organic solvents and detergents indicated that fractions known to be enriched in glycoproteins underwent more rapid degradation.

The high molecular weight components of liver plasma membranes and derivative fractions were more rapidly degraded than those of low molecular weight. Since glycoproteins predominated amongst the high molecular weight components of plasma membranes, the functional significance of this finding is discussed.
Isolation of Multiple Forms of DNA‐Dependent RNA Polymerase from Calf‐Liver Nuclei
Authors: Cacace Marcello G. , Nucci Roberto
Published: March 2005

Three species of DNA‐dependent RNA polymerase were separated from calf liver nuclei. The nuclei were obtained from up to a maximum of 1800 g of homogenized tissue by centrifugation at low speed and were successively cleaned by centrifugation in 2 M sucrose and 1 mM MgCl2. The purification procedure consisted of sonication at high ionic strength of the nuclear pellet, chromatin removal and ammonium sulphate precipitation of the solubilized activity. The three species were obtained by chromatography on a DEAE‐Sephadex column and have been designated form A(0) and form A, both insensitive to α‐amanitin, and form B, sensitive to this toxin. They could be distinguished according to their dependence on ionic strength and pH, requirement of metal ions, and template specificity. The first peak of activity is not present in preparations from calf thymus, and its characteristics are different from any other nuclear RNA polymerase activity separated by DEAE‐Sephadex chromatography. Its presence seems then to be correlated to the occurrence of tissue specificity of mammalian RNA polymerase.
Fluorescent Labels on Ribosomes
Authors: Pochon François , Ekert Bernard
Published: March 2005

70‐S ribosomes have three binding sites for the dimer of 1‐anilino‐8‐naphthalenesulfonate, a specific ligand for proteins. Synthesis of polyphenylalanine is not inhibited by the fixation of three dye molecules per ribosome. At low concentration of Mg2+, four and two molecules of dye can bind respectively to 50‐5 and 30‐S ribosomal particles and reassociation to functional ribosomes is always possible when the Mg2+ content is increased. These variations of the average number of binding sites are interpreted as being due to a conformational modification induced by Mg2+. Activity for protein synthesis and spectroscopic properties of ribosomes covalently bound to the carcinogen 7‐methylbenz[a]anthracene which is specific for ribosomal RNA Are Also described and polarisation studies of the ternary complex ribosome, mRNA, tRNA using a poly(A) labeled with the same compound indicates the mobility of the messenger in this complex.
The Effect of Insulin and Growth Hormone on the Flux of Tracer from Labelled Lactate in Perfused Rat Heart
Authors: Mowbray John , Ottaway James H.
Published: March 2005

l[2.14C]Lactate tracer was added at 30 min to the perfused rat‐heart preparation described in the previous paper. The specific activities of perfusate and tissue lactate and pyruvate were measured over the period up to 60 min.

Perfusate and heart lactate radioactivity equilibrated rapidly: not so perfusate and heart pyruvate.

No pyruvate was lost from the tissue: the sole precursor of perfusate pyruvate was lactate. In the presence of insulin all pyruvate taken up from perfusate was obligatorily reduced to lactate.

A proportion of pyruvate in the heart did not become labelled and there is good reason to believe this is not an artifact.

Rate parameters at different steps in pyruvate metabolism have been determined by computer modelling. Both hormones modify these parameters: in particular pyruvate dehydrogenase appears to undergo rapid 7‐fold activation.

The rate of exchange of label between lactate and intracellular labelled pyruvate was used to calculate the [NAD]/[NADH] ratios in the exchange compartment and in mitochondria. Both hormones reduced the ratio about 5 times by comparison with control. The results emphasise the disparity between perfusate lactate/pyruvate ratio and the redox potential of cardiac cytoplasm.

The data strongly support the compartmentization in more than one distinct cytoplasmic space of some glycolytic enzymes in cardiac tissue. The relationship of these “compartments” to heart morphology is briefly discussed.

Evidence is presented that growth hormone exerts a much greater rapid “insulin‐like” effect than has hitherto been suspected.
Formation of the Yeast Mitochondrial Membrane
Authors: Ibrahim Nader G. , Stuchell Robert N. , Beattie Diana S.
Published: March 2005

Amino acid incorporation in vitro by isolated yeast mitochondria could be supported by either an external ATP‐regenerating system or by the respiratory‐chain‐linked synthesis of ATP. However, the incorporation rates obtained with ATP and succinate were almost double those obtained with the ATP‐regenerating system and were inhibited by either antimycin A or oligomycin. In either system, addition of chloramphenicol, erythromycin or ethidium bromide caused an 80–90% inhibition of incorporation, actinomycin D and cordycepin caused a 20% inhibition, while rifamycin or cycloheximide had no effect.

The rate of amino acid incorporation in vitro by isolated mitochondria was not subject to glucose derepression. In contrast, the rate of cycloheximide‐insensitive protein synthesis into mitochondrial membranes in vivo was lowered over 90% in repressed cells. Analysis of the labeled mitochondrial membranes by sodium dodecylsulfate gel electrophoresis indicated that the products of mitochondrial protein synthesis were qualitatively identical, but quantitatively different, in glucose‐repressed and derepressed cells. For example, a two‐fold increase in labeling into both the two peaks of highest molecular weight as well as the peak of lowest molecular weight was observed in mitochondrial membranes obtained from repressed cells as compared to mitochondrial membranes obtained from partially and fully derepressed cells.

The rate of amino acid incorporation in vitro as well as the labeling of mitochondrial membranes in vivo was stimulated 50—100% in partially derepressed yeast cells which had been grown in medium containing chloramphenicol for three hours, washed, and allowed to grow for one hour in fresh medium without chloramphenicol. The stimulation of labeling in vivo resulted in a threefold increase of labeling into the peak of lowest molecular weight with a concomitant decrease of labeling into the peaks of high molecular weight suggesting that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins. Furthermore, growth of the cells in the presence of cycloheximide, which would result in a depletion of the pool of mitochondrial proteins synthesized in the cytoplasm, followed by growth in fresh medium without cycloheximide, resulted in a 50% decrease in the incorporation rate in vitro.
Binding Studies on Rabbit‐Muscle Phosphoglucose Isomerase
Authors: Bruch Peter , Schnackerz Klaus D. , Chirgwin John M. , Noltmann Ernst A.
Published: March 2005

Binding studies utilizing the techniques of gel filtration, rate dialysis and equilibrium dialysis yielded a value of 2.0 binding sites per molecule for either substrate or inhibitors like 6‐phospho‐gluconate and pyridoxal 5′‐phosphate to rabbit muscle phosphoglucose isomerase, thereby establishing that the enzyme has one catalytic site per subunit. For the substrates and 6‐phospho‐gluconate studies were performed at various pH values. For 6‐phosphogluconate, the dissociation constant was found to change dramatically with increasing pH, following a pattern analogous to that deduced from catalytic rate measurements. For the catalytic equilibrium mixture of glucose 6‐phosphate and fructose 6‐phosphate, the Kd showed excellent agreement with the Km values reported earlier. The binding studies are discussed in relation to the current concept of the catalytic mechanism.
The Localization of DNA Ligase in the Chromatin of Animal Cells
Authors: Gaziev Ajub I. , Kuzin Alexander M.
Published: March 2005

ATP‐dependent DNA ligase, which catalyses a covalent repair of phosphodiester breaks induced in DNA by DNAase I, was isolated from chromatin of bone marrow cells of rabbits and purified (more than 400 times against its original state). A major part of the chromatin DNA ligase represents an activated adenylate ligase · AMP complex.
The Dissociation of Escherichia coli Ribosomes
Authors: Ball L. Andrew , Johnson Peter M. , Walker Ian O.
Published: March 2005

A systematic study has been made of the dissociation into subunits of the ribosomes of Escherichia coli. By analytical ultracentrifugation, ribosomes were shown to be in dynamic reversible equilibrium with their subunits. This enabled a thermodynamic analysis to be made of the association‐dissociation reaction and of several factors which influence it: the concentration of monovalent and divalent cations, the presence of transfer RNA and supernatant factors, the pH, and hydrostatic pressure. The apparent change in free energy for the association in 10 mM sodium phosphate pH 7.0 was–17.4 kcal/mol. Association was accompanied by the net binding of magnesium ions or the net release of monovalent cations (potassium, sodium, or Tris ions). Circular dichroism measurements of ribosomes and their isolated subunits over a range of magnesium and potassium ion concentrations indicated that a change in the conformation of each subunit accompanied association. The change occurring in the 50‐S subunit was dependent on the presence of potassium ions, whereas the change in the 30‐S subunit was magnesium‐ion dependent. The conformational change in the 50‐S subunit was critical for the association‐dissociation reaction.

Under ionic conditions which lead to ribosome dissociation one molecule of transfer RNA was released per 70‐S ribosome. This was an independent reaction which did not influence the subunit association‐dissociation equilibrium. The release of transfer RNA was accompanied by the net release of four magnesium ions, and occurred with an apparent change in the free energy of 20.8 kcal/mol.
Heterogeneity of NADPH‐Dependent Aldehyde Reductase from Human and Rat Brain
Authors: Ris Margret M. , von Wartburg Jean‐Pierre
Published: March 2005

Four multiple molecular forms of NADPH‐dependent aldehyde reductase are isolated by ion‐exchange and gel chromatography from human brain. Using the same procedure two multiple forms of NADPH‐dependent aldehyde reductase are found in rat brain.

The molecular weight of two of the forms of human brain aldehyde reductase is 40000 and of the other two forms it is 44000.

Large differences with respect to substrate specificity with a wide range of aliphatic and aromatic aldehydes are observed between the multiple enzyme forms of each species.

All enzyme forms use NADPH as coenzyme; additionally one enzyme of each species can use NADH as well.

Classical inhibitors of aldehyde and alcohol dehydrogenase do not affect the activity of the multiple molecular forms of aldehyde reductase from human and rat brain, but psychoactive substances such as barbiturates and chlorpromazine as well as “biogenic” acids are inhibitory.
Étude spectroscopique de l'acide folique
Authors: Thiéry Corinne
Published: March 2005

A spectroscopic study of folic acid (AB), pterin (A) and para‐aminobenzamide (B), using absorption, fluorescence, phosphorescence and electron spin resonance techniques, indicates the occurrence of spectroscopic differences between the behavior of solution of folic acid AB and equimolar solution of (A + B).

The experimental evidence may be summarized as follows: a probable hypochromic effect on the highest energy bands, and a bathochromic shift (8 nm) over the whole absorption spectrum of AB, compared to that of (A + B); highly efficient quenching (more than 95%) of the fluorescence of components A and B in AB, compared to (A + B); disappearance of phosphorescent and paramagnetic signal of B in AB, but not in (A + B). The experiments are carried out on samples in aqueous solution at pH 5.6 (neutral, sparingly soluble, form of A and AB) and at pH 10.5 (ionic form of A and AB).

These spectroscopic differences, observed between solutions AB and (A + B), suggest important intramolecular interactions between the two chromophores A and B in the molecule AB.

A folded configuration, in which pterin A and the aromatic ring of B are held in proximity by several hydrogen bonds, seems in better agreement with spectroscopic data, than the generally accepted planar form, with hyperconjugation over the two main rings.
The Labilization of Hemoglobin by Cobalt
Authors: Schulman Herbert M. , Sidloi Rose , Martinez‐Medellin Jaime
Published: March 2005

To elucidate a mechanism for cobalt‐stimulated globin synthesis in rabbit reticulocytes, the cells were incubated with 1 mM CoCl2 containing 60Co. Although the cells incorporated cobalt irreversibly, evidence for the synthesis of cobalt‐protoporphyrin was not obtained. More than 90% of the cobalt recovered in the cell cytoplasma was bound to the globin moiety of hemoglobin. Similar results were obtained with erythrocytes and purified hemoglobin incubated with 60CoCl2.

By CM‐Sephadex chromatography and isoelectric focusing it was found that cobalt‐labelled hemoglobin differs slightly in charge from native hemoglobin, possessing a lower isoelectric point.

It was also demonstrated that the heme in cobalt‐labelled hemoglobin is less firmly bound to globin than the heme in the native protein, and that the purified cobalt‐treated protein can stimulate globin synthesis in rabbit reticulocytes.

It is postulated that in reticulocytes cobalt stimulates globin synthesis indirectly by labilizing intracellular hemoglobin.