bluuebunny/crossref_metadata_2025 huggingface dataset:
A Kinetic Study of the Energy‐Linked Influx of Ca2+ into Heart Mitochondria
Authors: CROMPTON Martin , SIGEL Erwin , SALZMANN Marianne , CARAFOLI Ernesto
Published: March 2005

The energy‐linked uptake of Ca2+ by heart mitochondria is inhibited by Mg2+ and this has been exploited to devise an inhibitor‐stop method which has permitted direct measurements of the kinetics of Ca2+ uptake. The initial rate of Ca2+ influx has also been measured directly using a Ca2+‐selective electrode. Half‐maximal rate of uptake is attained at 12–15 μM Ca2+ and the maximum velocity is about 1.4 nmol of Ca2+/mg of protein per s at 25°C. The rate versus Ca2+ ‐concentration curve is hyperbolic but becomes sigmoidal in the presence of Mg2+. Inorganic phosphate stimulates the initial rate of Ca2+ influx 2 to 4 times.
Derivatives of Guanosine Triphosphate with Ribose 2′‐Hydroxyl Substituents
Authors: HAMEL Ernest
Published: March 2005

This report describes the preparation of four methylated and phosphorylated derivatives of GTP, 2′‐O‐methylguanosine 5′‐triphosphate (PPP‐Me2′Guo), guanosine 2′‐monophosphate 5′‐triphosphate (PPP‐Guo‐2′P), 3′‐O‐methylguanosine 5′‐triphosphate (PPP‐Me3Guo), and guanosine 3′‐monophosphate 5′‐triphosphate (PPP‐Guo‐3′P) These compounds were compared to GTP in their ability to support reactions catalyzed by Escherichia coli initiation factor 2 (IF‐2), elongation factor Tu (EF‐Tu), and elongation factor G (EF‐G).

As with previously studied GTI analogues, the nucleotide specificities of IF‐2‐dependent N‐formylmethionylpuromycin formation and EF‐Tu‐dependent Ac‐Phe2‐tRNA formation were similar. There was little difference between the reactions supported by GTP, PPP‐Me 2′ Guo, PPP‐Me3′ Guo, and PPP‐Guo‐3′P, but PPP‐Guo‐2′P was a poor substrate with both enzymes.

A spectrum of activity was observed in EF‐G‐dependent formation of N‐acetylphenylalanyl‐phenylalanylpuromycin. While PPP‐Me2′ Guo was almost as effective as GTP in supporting translocation. PPP‐Guo‐2′P was a very poor substrate, having even less activity than guanosine 3′‐di‐phosphate 5′‐triphosphate. Intermediate activities were observed with PPP‐Me3′ Guo and PPP‐Guo‐3′P, the former nucleotide being more active than the latter.
Enzymic Reduction of Disulfide Bonds by Thioredoxin
Authors: HOLMGREN Arne , MORGAN Francis J.
Published: March 2005

The NADPH‐dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, α and β, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12μM thioredoxin and 0.1 μM thioredoxin reductase at pH 7 all disulfide bonds in the α subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the α subunit with thioredoxin followed by S‐carboxymethylation with iodo[2‐3H]acetic acid and analysis of tryptic peptides indicated that all S‐S bonds in the a subunit were surface oriented and equally reactive. The usefulness of thioredoxin reducide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.
Protein Degradation during Yeast Sporulation Enzyme and Cytochrome Patterns
Authors: BETZ Heinrich , WEISER Ulrike
Published: March 2005

The levels of several enzymes have been studied during sporulation of Saccharomyces cerevisia. The specific activities of ribonuclease and aminopeptidase I raised several‐fold after transfer of the cells to sporulation medium, whereas the specific activities of phosphofructokinase, glucose‐6‐phosphate dehydrogenase, tryptophan synthase and pyruvate decarboxylase were not significantly altered. The specific activities of NAD‐dependent glutamate dehydrogenase, isoeitrate lyase, malate dehydrogenase and fructose bisphosphatase all decreased from the onset of sporulation. The inactivation of these latter enzymes was inhibited by cycloheximide and by inhibitors of energy metabolism. Hexokinase, alcohol dehydrogenase and glutamate oxaloacetate transaminase were partially lost from the cells during the period of ascus maturation. None of the enzymic changes observed proved to be ‘sporulation‐specific’ in that it occurred exclusively in sporulating diploid yeast cells. Therefore it is postulated that the meiotic events and the metabolic changes required for ascospore formation are under separate genetic control in this organism.

During sporulation, the cellular content of cytochromes b, c, and aa3 was reduced to 20% or less of that present in vegetative derepressed cells. Since the relative percentage of total to cycloheximide‐insensitive mitochondrial protein synthesis was not significantly altered throughout sporulation, and the pattern of mitochondrially synthesized polypeptides was rather similar both in vegetative and in sporulating cells, it appeared that not not only degradation but also synthesis and therefore turnover of the mitochondrially coded polypeptides of cytochromes b and aa3 took place during sporulation. The activity ratio of cytochrome c oxidase to F1‐ATPase in submitochondrial particles isolated from vegetative cells and. from purified asci was almost identical. This indicates that the loss of membrane‐bound mitochondrial cytochronies during sporulation is probably due to a nonselective degradation of inner mitochondrial membrane proteins.
The Binding Site of Protein L1 on 23‐S Ribosomal RNA of Escherichia coli
Authors: SLOOF Paul , GARRETT Roger , KROL Alain , BRANLANT Christians
Published: March 2005

Ribonucleoproteins were prepared by ribonuclease digestion of a reconstituted complex of ribosomal protein L 1 and 23‐S RNA frons Escherichia coli. Three main ribonucleoproteins were identified. The largest was only obtained in an impure state at low ribonuclease concentrations, whereas the two smaller ones, which were difficult to separate from one another electrophoretically, were stable over a range of enzyme concentrations.

The two smaller ribonucleoproteins yielded a total of 13 RNA subfragments that were judged to be homogeneous electrophoretically. The latter were characterized for molecular weight and the subfragment composition of each of these ribonucleoproteins was established. Furthermore, the subfragments were shown to be maintained together in each ribonucleoprotein by RNA‐RNA interactions

The primary and specific binding site of protein L1 was localized on one continuous RNA subfragment of about 110 nucleotides in length by two newly developed binding methods.
The Binding Site of Protein L1 on 23‐S Ribosomal RNA of Escherichia coil
Authors: BRANLANT Christiane , KROL Alain , SRIWIDADA Johannes , EBEL Jean‐Pierre , SLOOF Paul , GARRETT Roger A.
Published: March 2005

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstituted complexes of ribosomal protein L1 and 23‐S RNA From Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with T 1 ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th nucleotides from the 3′ end of‐ the 23‐S RNA.

Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5′ end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3′ end of the region.

The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L 1 most strongly protects a continuous section of‐ 115 nucleotides at the 5′ end of the main RNA region.

Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23‐S RNA.
Studies on 3‐Deoxy‐d‐arabinoheptulosonate‐7‐phosphate Synthetase(phe) from Escherichia coli K12
Authors: SIMPSON Richard J. , DAVIDSON Barrie E.
Published: March 2005

Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the α‐carboxyl and α‐amino groups of phenylalanine for inhibition of 3‐deoxy‐d‐arabinoheptulosonate‐7‐phosphate synthetase(phe) activity. Replacement of the α‐H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono‐substituted fluoro, chloro and hydroxy phenylalanines. ‐d‐Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity.

Circular dichroism studies indicated that the native enzyme possesses approximately 26%α‐helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in the interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremely hydrophobic environment to one less hydrophobic.

K
d for the binding of phenylalanine to the enzyme was determined spectrophotometrically to he 75 μM.

Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue nay be implicated in the catalytic activity of the enzyme.
Subunit Structure of Simian‐Virus‐40 Minichromosome
Authors: BELLARD Maria , OUDET Pierre , GERMOND Jacques‐Edouard , CHAMBON Pierre
Published: March 2005

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 ± 2 nucleosomes, each containing 190‐200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190‐200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a ‘core’ containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.
Biochemical and Electron‐Microscopic Evidence that the Subunit Structure of Chinese‐Hamster‐Ovary Interphase Chromatin Is Conserved in Mitotic Chromosomes
Authors: COMPTON J. Lee , HANCOCK Ronald , OUDET Pierre , CHAMBON Pierre
Published: March 2005

Biochemical and electron microscopic studies demonstrate that the subunit structure Of Chinese hamster ovary cell interphase chromatin is conserved in mitotic chromosomes. Digestion of purified chromosomes or nuclei with micrococcal nuclease produces DNA in discrete size classes, as visualized by polyacrylamide gel electrophoresis, which are common to the two materials. Early in digestion the DNA fragments are integral multiples of a monomer approximately 177 base pairs in length, whereas after extensive digestion the remaining DNA fragments migrate ahead of the monomer position. The size of the repeating DNA unit was confirmed as being smaller than that produced by micrococcal nuclease digestion of rat liver nuclei by direct comparison. Electron microscopy of partially unravelled chromosomes at low ionic strength shows tightly packed spheres (nucleosomes) approximately 12 nm in diameter which are often arranged as linear chains. Chromosomal material prepared for electron microscopy after varying extents of micrococcal nuclease digestion is composed of fragments containing progressively fewer nucleosomes, which parallels the loss of high DNA multimer hands in gel electrophoresis. Material unravelled from chromosomes in the presence of NaCl consists of nucleosomes packed in a different configuration which suggests the origin of higher order structures in chromosomes.
Purification and Identification of a Human‐Serum DNA‐Binding Protein Associated with Malignant Diseases
Authors: PARSONS Robert G. , HOCH James A.
Published: March 2005

A malignancy‐associated human serum DNA‐binding protein (C3DP protein), which was previously detected using dodecylsulfate gel electrophoresis, has been purified and characterized. This protein was isolated from human fetal cord serum by DNA‐cellulose affinity chromatography, ammonium sulfate fractionations, DEAE‐cellulose chromatography, and Sephadex gel filtration. The molecular weight of purified C3DP protein has been shown to be 135000 by ultracentrifugation, gel filtration, and dodecylsulfate gel electrophoresis. Dodecylsulfate gel electrophoresis following disulfide bond reduction has revealed that this protein is composed of three subunits having molecular weights of 74000, 40000 and 22000. Carbohydrate has been demonstrated to be attached to the 74000 and 22000 molecular weight components. Immunochemical studies have revealed that the C3DP protein is a fragment of human complement component C3, which closely resembles C3c.
Separation of Malate Dehydrogenase Isoenzymes by Affinity Chromatography on 5′‐AMP‐Sepharose
Authors: WALK Rüdiger‐A. , HOCK Bertold
Published: March 2005

The mitochondrial and glyoxysomal isoenzymes of malate dehydrogenase (EC 1.1.1.37) from watermelon cotyledons and the mitochondrial isoenzyme from pig heart adsorbed reversibly to 5′‐AMP‐Sepharose. They were specifically eluted with low concentrations of NADH rather than by NAD. In contrast, the cytoplasmic isoenzymes showed no affinity to the matrix‐bound ligand. These binding properties are discussed in terms of structural and regulatory differences of the particulate and soluble malate dehydrogenase isoenzymes. Affinity chromatography on 5′‐AMP‐Sepharose significantly improved the purification of the particulate malate dehydrogenase isoenzymes with respect to homogeneity, yield, and the number of purification steps. In the case of the glyoxysomal isoenzyme it was the essential procedure to obtain complete purification of the enzyme.
Evidence for the Nucleotide Sequence of 5‐S rRNA from the Flowering Plant Secale cereale (Rye)
Authors: PAYNE Peter I. , DYER Tristan A.
Published: March 2005

Evidence for the sequence of rye 5‐S rRNA was derived from the analysis of partial and complete enzymic digests of the 32P‐labelled molecule. The probable sequence of 5‐S rRNA from four other flowering plants was deduced by aligning, with the homologous sequences in the rye 5‐S rRNA, oligonucleotides produced by T1 and pancreatic A ribonuclease digestion. The most dissimilar differed in only seven positions. From a comparison of the sequence of rye 5‐S rRNA with those known for other types of organism, it was possible to distinguish some structural features of the molecule which are common to all of them. Also, information was obtained about the possible phylogenetic relationship of the flowering plants to other organisms whose 5‐S rRNA has been sequenced.
Five‐Coordinate Iron‐Porphyrin as a Model for the Active Site of Hemoproteins
Authors: MOMENTEAU Michel , ROUGÉE Michel , LOOCK Bernard
Published: March 2005

Preparation of iron(III)‐deuteroporphyrin 6(7)‐methyl ester, 7(6)‐(histidine methyl ester) by coupling histidine methyl ester to deuterohemin has been performed using the mixed carboxylic/carbonic‐acid‐anhydride method. This compound, which is very soluble in various organic solvents, can be considered as a prosthetic group model for the active site of five‐coordinate hemoproteins. In the oxidized state a basic, a neutral or an acid form can be isolated. The basic and acid forms are monomeric at all concentrations. The neutral form is found dimeric in concentrated solutions while it is monomeric at low concentrations. The coordination state of iron in these various species is investigated. The neutral form reacts with ligands, such as nitrogenous organic bases, leading to six‐coordinate well‐known hemichromes which exhibit low‐spin electron spin resonance (ESR) spectra. The reaction of anionic ligands, such as F−, CN−, NO2− and N3−, with the neutral form model leads to unsymmetrical six‐coordinate complexes whose optical and ESR spectra are similar to those of synthetic deuteromyoglobin.

In benzene, toluene or dichloromethane solutions iron(II)‐deuteroporphyrin 6(7)‐methyl ester, 7(6)‐histidine methyl ester), obtained from ferric forms by heterogeneous reduction with aqueous dithionite, exhibits an optical spectrum characteristic of a five‐coordinate high‐spin ferrous complex. At low temperature important spectral modifications are observed indicating a dimeric association. At room temperature it binds one nitrogenous base molecule leading to the well‐known hemochrome. It reacts also with carbon monoxide with a very high affinity constant (4.5 × 108 M−1), comparable to that of the isolated human hemoglobin α and β chains, but much higher than the values reported for other various models, suggesting that the side‐chain length bearing the fifth ligand may have an important influence upon the reactivity of the sixth position of the iron ion. At low temperature in toluene or dichloromethane, this compound reversibly binds oxygen without oxidation of the iron ion while oxidation occurs at room temperature. The significance of these results is discussed in relation with the local environment, the electronic nature of the base and the immobilization of the heme group in hemoproteins.
A New Plant‐Type Ferredoxin from Halobacteria
Authors: KERSCHER Lorenz , OESTERHELT Dieter , CAMMACK Richard , HALL David O.
Published: March 2005

A stable, 2Fe‐type ferredoxin has been prepared from Halobacterium halobium and purified by chromatography. A similar ferredoxin was also found in three other Halobacteria. The ferredoxin is present in large amounts—about 1% of the total soluble protein. From amino acid composition a molecular weight of 14800 ± 200 was calculated. The ferredoxin was found to contain two atoms each of iron and sulphide. The midpoint redox potential of the protein is about—345 mV. The electron paramagnetic resonance spectrum of the reduced form shows much similarity to plant and algal ferredoxins with gx= 1.90, gy= 1.97 and gz= 2.07. The same similarity is observed in the optical absorption, optical rotatory dispersion and circular dichroism spectra. However it does not seem to mediate electron transport in the NADP‐photoreduction system of chloroplasts. Extracts of the bacterial cells catalyze the reduction of the ferredoxin by NADH.
Quantitative Determination of N‐Acetylglucosamine Residues at the Non‐reducing Ends of Peptidoglycan Chains by Enzymic Attachment of [14C]‐d‐Galactose
Authors: SCHINDLER Melvin , MIRELMAN David , SCHWARZ Uli
Published: March 2005

The ability of human milk galactosyltransferase to attach d‐galactose residues quantitatively to the C‐4 of N‐acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rodshaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical ‘mini’ cells had chain lengths similar to those of the isogenic rod‐like cells.
Free 3′‐OH Group of the Terminal Adenosine of the tRNA Molecule Is Essential for the Synthesis in vitro of Guanosine Tetraphosphate and Pentaphosphate in a Ribosomal System from Escherichia coli
Authors: SPRINZL Mathias , RICHTER Dietmar
Published: March 2005

tRNAphe from yeast modified at the 3′‐terminal adenosine residue or missing part of its CpCpA end was investigated for its ability to participate in the synthesis of guanosine tetraphosphate and pentaphosphate using Escherichia coli ribosomes. Only tRNA containing complete CpCpA end and an intact ribose residue in the terminal adenosine was active in this process. The absence of the 2′‐hydroxyl group of the terminal adenosine does not influence the reaction, but the 3′‐hydroxyl group is essential and cannot be replaced by an amino group or a hydrogen atom. tRNA in which the 3′‐terminal adenosine is replaced by formycin is active in the induction of guanosine tetraphosphate and pentaphosphate synthesis, but the reaction proceeds relatively slowly.
A Complex Control Circuit
Authors: THOMAS René , GATHOYE Anne‐Marie , LAMBERT Lucie
Published: March 2005

Temperate bacteriophages can display in a stable way two essentially different behaviours. In the immune state, a gene (cI) produces a repressor which prevents expression of all the other viral genes; in the non‐immune state the typically viral functions are expressed. The choice between the two pathways and the establishment of one of them have much in common with cell determination and differentiation. This choice depends on a complex control system, in fact one of the most intricate nets of regulation known in some detail. Our paper provides a formal description and partial analysis of this regulatory net. It is shown that even for relatively simple known models, this kind of analysis uncovers predictions which had previously remained hidden. Some of these predictions were checked experimentally.

The experimental part chiefly deals with the efficiency of lysogenization by thermoinducible lambda phage carrying mutations in one or more of the regulatory genes, N, cro and cII. Although N− mutations are widely known for preventing efficient integration, and both N− and cII mutations for preventing efficient establishment of immunity, it is shown that, as predicted by a simple model, both N− and cII− phage efficiently lysogenize at low temperature if they are in addition cro−. In contrast with λ N− cro+, λ N− cro− is not propagated as a plasmid at low temperature, precisely because it establishes immunity too efficiently.

Genetic control circuits are described in terms of sets of logic equations, which relate the state of expression of genes or of chemical reactions (functions) to input (genetic and environmental) variables and to the presence of gene and reaction products (internal, or memorization variables). From the set of equations, one derives a matrix which shows the stable stationary states (if any) of the system, and from which one can derive the pathways (temporal sequences of states) consistent with the model. This kind of analysis is complementary to the more widely used analysis based on differential equations; it allows one to analyze in less detail more complex systems. The language might be used as well, mutatis mutandis, in fields very different from genetics.

The last part of the discussion deals with the role of positive feedback loops in our specific problem (establishment and maintenance of immunity in temperate bacteriophages) and in developmental genetics in general. As a generalization of an old idea, it is suggested that cell determination (for a given character) depends on a set of genes whose interaction constitutes a positive feedback loop. Such a system has two stable stationary states: which one is chosen will usually depend on additional controls grafted on the loop.
Small‐Angle X‐Ray Studies of the Fab and Fc Fragments from the Human Immunoglobulin Molecule Kol
Authors: PILZ Ingrid , SCHWARZ Erika , PALM Walter
Published: March 2005

The conformation of the Fab and Fc fragments from the human immunoglobulin molecule Kol [IgG I, χ2γ2, Gm(f)+] was studied by small‐angle X‐ray scattering in solution. The fragments were studied in 0.02 M Tris‐HCI buffer. For the Fab fragment the radius of gyration was found to be 3.15 ± 0.15 nm, the volume to be 75 ± 8 nm3. For the Fc fragment the respective values were 3.15 ± 0.15 nm for the radius of gyration and 91 ± 8 nm3 for the volume. A large number of models were calculated for both fragments to find models which fit these data and have the same scattering curve. The models with the best agreement were compared with the models found for the crystalline state by crystal X‐ray studies.
Esterolytic Activities of Rat Intestinal Mucosa
Authors: FERNANDEZ‐LOPEZ Victor , SERRERO Ginette , NÉGREL Raymond , AILHAUD Gérard
Published: March 2005

A glycerol‐ester hydrolase from rat intestinal cells has been purified using chromatography on carboxyhexanoyl‐Sepharose‐glyceryldioctanoate and preparative gel electrophoresis. The enzyme gives a single band by analytical gel electrophoresis; it is a monomer of molecular weight 68000. The optimum pH for its action on glyceryl tributyrate is between 8.0 and 8.5; the activation energy was calculated to be 8.7 kcal × mol−1 (36.4 kJ/mol). Its substrate specificity is mainly directed against esters of glycerol and of primary monoalcohols. Similarly to pancreatic lipase but contrary to liver esterase, it is inhibited by bile salts; relief of this inhibition by colipase is only observed for pancreatic lipase. The possible role of the glycerol‐ester hydrolase in the absorption of short and of medium chain triglycerides is discussed.
Proton Equilibria in the Binding of Zn2+ and of Methylmercuric Iodide to Papain
Authors: SLUYTERMAN Lamoraal A. Æ. , WIJDENES Jacob
Published: March 2005

The proton liberation on the binding of zinc chloride and methylmercuric iodide to the (essential) thiol group of papain has been examined as a function of pH. This was carried out by (a) direct titration of the protons on the addition of the metal compound to active papain and (b) measurement of the extent of inhibition of enzyme activity by the metal compound as a function of pH.

It was found that in the neutral pH range the thiol group or the neighbouring imidazole group in the free enzyme carries one proton, at low pH both groups do so, whereas at high pH neither group carries a proton. The pK values of the free enzyme that govern the proton release, 4.2 and 8.5, correspond to those that govern overall activity. Both from the experiments with methylmercuric iodide and from fluorescence measurements of methylmercuric papain, it was established that the imidazole group in the latter compound exhibits a pK of 5.4. Taking recent data into account, it was considered that the ion pair of thiolate anion and imidazolium cation, proposed by Polgar, is the best approximation to describe the charge distribution in the active centre and to explain the reaction mechanism.
Catalase Biosynthesis in Yeast: Formation of Catalase A and Catalase T during Oxygen Adaptation of Saccharomyces cerevisiae
Authors: ZIMNIAK Piotr , HARTTER Engelbert , WOLOSZCZUK Wolfgang , RUIS Helmut
Published: March 2005

Catalase A from Saccharomyces cerevisiae and its biosynthetic precursors can specifically be immunoprecipitated from extracts obtained from yeast cells grown in the presence of l‐[3H]leucine or 59FeCl3. The enzyme and its precursors recognized by a specific antiserum are absent from anaerobic cells. During oxygen adaptation of yeast pre‐grown on 0.3% glucose under anaerobic conditions catalase A is formed via a heme‐less precursor, probably the apomonomer of the protein, and a heme‐containing intermediate. When cells are grown in the presence of Tween 80 the amount of catalase A, but not of catalase T, increases 4‐fold. Comparison of the mode of synthesis of catalase T and A shows that no precursor‐product relationship exists between the two proteins.
Kinetics of the Interaction between Aspartic Aminotransferase and Anions
Authors: GIANNINI Ivo , BARONCELLI Vittorio , BOCCALON Gianfranco , FASELLA Paolo
Published: March 2005

The kinetics of the interaction between deionized supernatant aspartic aminotransferase and various anions (cacodylate, phosphate and chloride) were studied by the temperature‐jump technique.

The anion concentration in the range covered by our experiments does not affect the transamination rate. On the other hand the conformational transition, recently observed at the active site of the enzyme, is hindered by an excess of anions.

A single relaxation effect was observed at the enzyme chromophore wavelength in systems containing the aldimine form of the enzyme and the above anions. It is shown that this effect corresponds to the protonation of the chromophore. The relaxation times were of about 10 μs with phosphate, 20–100 μs with cacodylate and 1–2 ms with chloride. The pH and concentration dependence of this effect were studied. The fits of experimental data to a rate equations for various models were tested by a X2 analysis. The best fit was obtained with models where anions bind rapidly to a site close to the chromophore, so that the pK of the chromophore is affected by anions binding. The rate of the observed relaxation considerably increased when the anion has buffering capacities; this indicates, in the case of cacodylate and phosphate, that the acidic component of the buffer directly exchanges a proton with the enzyme chromophore.
An Analysis on the Slope of Scatchard Plots
Authors: HENIS Yoav I. , LEVITZKI Alexander
Published: March 2005

The Scatchard plot, [X]b/[E]t[X] versus [X]b/[E]t where [X]b denotes the concentration of bound ligand, [E]t the total concentration of the binding protein and [X] the free ligand concentration, was designed originally for plotting data of ligand binding to a macromolecule possessing identical non‐interacting ligand binding sites. However, the plot is used for describing cooperative binding processes. In such cases, the slope of the Scatchard plot is not then equal to minus the intrinsic association constant any more. The meaning of the slope in such cases is a complex function of the binding parameters and its exact interpretation depends on the particular model used to analyze the binding data. In this communication, the meaning of the slope of the Scatchard plot is analyzed in terms of the different allosteric models.
Metabolism of γ‐Glutamyl Amino Acids and Peptides in Mouse Liver and Kidney in vivo
Authors: ORLOWSKI Marian , WILK Sherwin
Published: March 2005

The metabolism in vivo of γ‐glutamyl amino acids and peptides was studied in the mouse after administration of loading doses of l‐γ‐glutamyl‐2‐aminobutyrate and several other γ‐glutamyl compounds, including glutathione. A great and rapid accumulation of glutamate, glutamine, aspartate and pyrrolidone carboxylate was observed in the kidney. Similarly, after administration of a tracer dose of l‐γ‐[14C]glutamyl‐l‐2‐aminobutyrate a rapid incorporation of label into kidney glutamate, glutamine and aspartate was found. These results suggest that both the hydrolytic and γ‐glutamyl transfer reactions catalyzed by γ‐glutamyl transpeptidase are active in the renal handling of γ‐glutamyl compounds.

Indirect evidence was obtained that l‐γ‐glutamyl‐2‐aminobutyrate is partially taken up by the kidney cell in an intact form.

In contrast to the kidney, administration of several γ‐glutamyl derivatives did not cause an increase in liver glutamate, glutamine and pyrrolidone carboxylate. After administration of l‐γ‐glutamyl‐2‐aminobutyrate only a slight increase in liver aspartate and pyrrolidone carboxylate was observed. Experiments with l‐γ‐[14C]glutamyl‐l‐2‐aminobutyrate suggest that this derivative is largely first degraded to its component amino acids (probably in the kidney) before entering into the metabolism of the liver cell.

γ‐Glutamyl transpeptidase may function in the metabolism and transport of glutathione and other γ‐glutamyl compounds in a manner analogous to the function of dipeptidases and disaccharidases in the metabolism and transport of dipeptides and disaccharides respectively.
Effect of the Immediate Environment on the Reactivity of the Essential—SH Group of Papain
Authors: HALÁSZ Piroska , POLGÁR László
Published: March 2005

The effect of the microenvironment on the reactivity of the essential—SH group of papain was studied by alkylation with methyl iodide and with the more polar iodoacetamide. Rate and activation parameters for these reactions were determined with two forms of the—SH group: the free mercaptide ion at pH 10.0, and the mercaptide‐imidazolium ion‐pair at pH 5.5.

The ion‐pair of papain reacts with methyl iodide at a rate 1470 times less than that of thiolsubtilisin. This suprising difference between the reactivities of the two enzymes suggests that in contrast to thiolsubtilisin, where a non‐polar environment enhances the rate, in the case of papain a more polar environment somewhat inhibits the reaction with the non‐polar methyl iodide. The positive activation entropy for the papain reaction may indicate an ‘ordered’ structure of bound water around the sulfur atom.

The high rate and the low activation entropy (organized transition state) of the reaction of papain with iodoacetamide can be explained in terms of hydrogen‐bond formation between the enzyme and the amide group of the alkylating agent.
Terminal Riboadenylate Transferase from Escherichia coli
Authors: SANO Hiroshi , FEIX Günter
Published: March 2005

Catalytic properties of terminal riboadenylate transferase from Escherichia coli and the products of the enzymic reaction were investigated. The kinetic analysis revealed that the reaction obeys the sequential ordered bi‐bi mechanism. The application of conditions elaborated in this study resulted in the synthesis of products of defined size and efficient primer utilization. The tRNA(rA)n obtained was a good template for the synthesis of complementary DNA with réverse transcriptase.
Proton‐Nuclear‐Magnetic‐Resonance Study of the Conformation of Neurotoxin II from Middle‐Asian Cobra (Naja naja oxiana) Venom
Authors: ARSENIEV Aleksandr S. , BALASHOVA Tamara A. , UTKIN Yurii N. , TSETLIN Viktor I. , BYSTROV Vladimir F. , IVANOV Vadim T. , OVCHINNIKOV Yurii A.
Published: March 2005

A proton nuclear magnetic resonance (NMR) study at 100 and 300 MHz of neurotoxin II from the venom of Middle‐Asian cobra Naja naja oxiana has been performed in 2H2O and H2O solutions. By means of chemical modification and double resonance all the aromatic residue resonances have been assigned. From the NMR titration curves, pK values of histidine 4 and histidine 31 residues have been determined. For one of the two neighbouring tryptophan residues pH dependence (in the 2–8‐pH range) of the chemical shifts of indole protons has been revealed. According to the different sensitivity of the linewidth of indole NH resonances to pH in H2O solution, the accessibility of each of the tryptophan residues has been estimated. Temperature dependence has been observed for the linewidth of the aromatic resonances of the tyrosine 24 residue. Deuterium exchange rates have been measured for amide protons as well as for C(2)H histidine resonances.

The NMR data obtained have allowed the conclusions to be made that the two histidine residues and one of the tryptophan residues should be localized on the surface of the protein globule, that arginine residues should be present in the environment of histidine 4, that histidine 31 and the buried tryptophan are possibly localized in close spatial proximity and that the side chain of tyrosine 24 is buried within the protein globule.
The Effect of Initiation Factor IF‐1 on the Dissociation of 70‐S Ribosomes of Escherichia coli
Authors: NAAKTGEBOREN Nico , ROOBOL Kees , VOORMA Harry O.
Published: March 2005

By means of exchange studies, in which 3H‐labelled 50‐S subunits and unlabelled 70‐S ribosomes from Escherichia coli MRE 600 were used, it has been demonstrated that the 30‐S subunit is the only target for IF‐3 in the dissociation of 70‐S ribosomes. The interference of IF‐3 with the dynamic equilibrium of 70‐S ⇌ 50‐S + 30‐S occurs by binding of the factor to the 30‐S subunit. The 30‐S. IF‐3 complex in impaired in the association reaction, which implies that IF‐3 is acting as an anti‐association factor.

The action of IF‐1 is two‐fold. Firstly IF‐1 increases the rate of exchange of the ribosomal sub‐particles in the 70‐S ribosome without changing the position of the equilibrium. Thus the spontaneous equilibrium is attained more rapidly in the presence of IF‐1. This kinetic effect of IF‐1 is also demonstrated in the IF‐3‐mediated dissociation of 70‐S ribosomes. Secondly IF‐1 is able to increase the IF‐3‐mediated dissociation. It seems likely that the explanation for the latter phenomenon must be sought in the binding of IF‐1 to 70‐S ribosomes, resulting in a loosening of the ribosome structure, as well as to 30‐S. IF‐3 complex, thereby slowing down the association reaction of the subunits.
Kinetics and Regulation of Sarcoplasmic Reticulum ATPase
Authors: DUPONT Yves
Published: March 2005

The measurement of ATP binding to the sarcoplasmic reticulum membrane reveals that the calcium pump possesses one high affinity (Kd= 2–3 μM) site. Competition with substrate analogs show the high specificity of that site. At high ATP concentration another class of site can be detected with a much higher dissociation constant (Kd∼ 500 μM). This class of sites is of low specificity and ATP is easily displaced by other polyphosphates.

The steady state rate of ATP cleavage is measured in the presence of ATP analogs. It is shown that the catalysis is due to the high affinity site. The activation of the hydrolysis rate at high substrate concentration may be related to the effect of binding of ATP to the weak sites. The effect of ATP analogs for various ATP concentration supports this hypothesis.
Ligand‐Dependent Bohr Effect of Chironomus Hemoglobins
Authors: STEFFENS Guy , BUSE Gerhard , WOLLMER Axel
Published: March 2005

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation.

For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2‐liganded and CO‐liganded states of hemoglobin III.

Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi: here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively.

On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine‐E11, obstructing the movement of the heme‐iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved.

The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.
Photooxidation of Human Serum Albumin and Its Complex with Bilirubin
Authors: PEDERSEN Anders Overgaard , SCHØNHEYDER Fritz , BRODERSEN Rolf
Published: March 2005

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen.

Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non‐oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected.

It is considered possible that the fast, anaerobic process is operative in phototherapy of hyper‐bilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.
Adenosine 3′:5′‐Monophosphate Metabolism and Turnover in Dog Thyroid Slices
Authors: VAN SANDE Jacqueline , SWILLENS Stéphane , DUMONT Jacques E.
Published: March 2005

The metabolism and turnover of adenosine 3′: 5′‐monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half‐life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effects on cyclic AMP disappearance. The methodology of the calculation of half‐life and turnover from such data is discussed.
Stereospecificity of Phenylyruvate Tautomerase
Authors: RÉTEY Jànos , BARTLV Knut , RIPP Engelbert , HULL William E.
Published: March 2005

By 1H nuclear magnetic resonance spectroscopy it has been shown that phenylpyruvate tautomerase from beef kidney catalyses the stereospecific exchange of one of the enantiotopic 3‐H atoms in the side‐chain of the substrate with solvent protons, the rate of spontaneous exchange being slow under physiological conditions.

Using monodeuterated phenylpyruvate of known absolute configuration it has been shown that the tautomerase removes specifically the 3‐pro‐R hydrogen atom of the substrate.

The circular dichroism spectra of the two enantiomeric 3‐phenyl‐[3‐2H]pyruvates have been determined.

Some implications of these findings for the investigation of the metabolism of aromatic amino acids are discussed.
Preparation and Coenzymic Activity of Soluble Polyethyleneimine‐Bound NADP+ Derivatives
Authors: ZAPPELLI Piergiorgio , PAPPA Rosario , ROSSODIVITA Antonio , RE Luciano
Published: March 2005

Alkylation at N‐1 of the NADP+ adenine ring with 3,4‐epoxybutanoic acid gave 1‐(2‐hydroxy‐3‐carboxypropyl)‐NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6‐(2‐hydroxy‐3‐carboxypropyl)‐NADP+. On the other hand, bromination at C‐8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3‐mercaptopropionic acid, gave 8‐(2‐carboxyethylthio)‐NADP+. Carbodiimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues.

The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co‐enzymically active with yeast glucose‐6‐phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiencies with the three enzymes of the N‐1 derivatives to the one of the corresponding N6 and C‐8 analogues, the order of activity was N‐1 > N6 > C‐8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.
Purification and Properties of Blood‐Group‐Specific Lectins fromVicia cracca
Authors: RÜDIGER Harold
Published: March 2005

The lectins from the seeds ofVicia craccareact specifically with human blood group A erythrocytes. They were purified by affinity chromatography on an adsorbent containing matrix‐boundN‐acyl‐d‐galactosamine. By a continuous pH gradient the lectins could be separated into two fractions each of which was shown to consist of several agglutinating species.

The behaviour of both fractions in affinity chromatography was paralleled by the pH dependence of the interaction with the hapten sugarN‐acetyl‐d‐galactosamine. Both lectin fractions have the same molecular (125000) and subunit (33000) weights, display the same pH dependence of their titre against A1erythrocytes, and bind toN‐acetyl‐d‐galactosamine at pH 8 with the same constant of about 6 × 103M−1.
RNA Polymerase and RNA Synthesis in the Silk Glands of the Silkworm Bombyx mori
Authors: SRIDHARA S. , PORTIER Marie‐madeleine , DAILLIE Jacques
Published: March 2005

Total RNA polymerase activity, as well as the proportion of a‐amanitin‐sensitive and resistant during activity, have been measured in the posterior silk glands of the silkworm as a function of growth the fifth larval instar. During the first 5 days, termed the growth phase, the total enzyme activity and particularly the portion that is α‐amanitin‐resistant increases to reach a peak value and thereafter declines during the secretory phase. Much of the enzyme remains firmly bound and insoluble. Heparin not only does not inhibit this insoluble and probably chromatin‐bound activity which would indicate lack of initiation, but it enhances the activity. A large proportion of newly transcribed RNA is released from the transcription complex.

The synthesis of RNA has been studied both qualitatively and quantitatively during the same period. RNA synthesis becomes important on the second day of the fifth instar, as does the RNA polymerase, and stays at a high level for several more days. The results from these studies as well as those with incorporation of 32P indicate interference of varying precursor pools in quantitatively measured RNA synthesis. However, RNA content as well as RNA synthesis in vitro show a close correlation with RNA polymerase activity. The labeled RNAs extracted at different days of the fifth instar have been fractionated on sucrose gradients; this demonstrated that the predominant product of RNA synthesis, as followed by [3H]uridine incorporation at short time intervals, is 45‐S preribosomal RNA and 4‐5‐S RNA. This 45‐S RNA is transformed to 19‐S and 30‐S ribosomal RNA as time progresses or after a chase with unlabeled uridine and/or actinomycin D. There also exists a component heavier than 45 S which is fairly rapidly labeled to a small extent.