bluuebunny/crossref_metadata_2025 huggingface dataset:
Some Properties of “Light” Cytoplasmic Ribonucleoprotein of Mouse Plasmacytoma
Authors: GRECHKO Vernata V. , SACCHAROVA Natalia K. , GORJUNOVA Ludmila E.
Published: March 2005

[5‐3H]Uridine incorporation into ribosomes and other postribosomal ribonucleoprotein particles has been determined in mouse plasmocytoma MOPC406 cytoplasm after cell wall disruption. It has been shown that a large part of the radioactivity after 40 to 90‐min incubation is localized in the postribosomal zone of the gradient. The question arises whether these particles are the result of nuclear leakage during cell disintegration or whether they are native to the cytoplasm. To clarify this we have compared the following characteristics of cytoplasmic and nuclear ribonucleoprotein particles: (a) sensitivity to sodium deoxycholate; (b) sedimentation patterns in sucrose gradients; (c) buoyant density values in CsCl gradient; (d) dynamics of precursors incorporation into cytoplasmic ribonucleoprotein particles RNA.

All these results have been considered as supporting the cytoplasmic origin of cytoplasmic postribosomal ribonucleoprotein particles.
Kinetics of the Interaction between Pig‐Plasma Benzylamine Oxidase and Various Monoamines
Authors: LINDSTRÖM Anders , OLSSON Bengt , PETTERSSON Gösta , SZYMANSKA Jadwiga
Published: March 2005

The kinetics of interaction between pig‐plasma benzylamine oxidase and a series of amine substrates have been investigated by conventional initial‐velocity studies, and by determination of transient rates of aldehyde formation and reduction of the enzymatic 470‐nm chromophore using stopped‐flow techniques. Kinetic parameters for the various processes and different substrates are reported.

The results obtained provide evidence that reduction of the enzyme by different monoamines leads to the formation of a common reduced enzymatic intermediate, reoxidation of which appears to be rate‐limiting in all the reactions examined. Km‐values for the different substrates are inversely proportional to second‐order rate constants for the corresponding reduction process, and exhibit a striking correlation to the base strength of the respective amines. The latter observation may suggest that benzylamine oxidase acts exclusively on the non‐protonated form of amine substrates.
Effects of Sodium Chloride and Pancreatic Ribonuclease on the Rat‐Brain Nuclear Particles: The Fate of the Protein Moiety
Authors: STÉVENIN James , JACOB Monique
Published: March 2005

Previous results on RNA‐labelled nuclear particles were compatible with a folded ribonucleo‐protein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vivo; they were prepared by sonication of nuclei.

1. Proteins were released progressively from the particles (ϱ= 1.39 g/ml) as shown by the accumulation of material of ϱ= 1.31–1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high‐density ribonucleoproteins were formed.

2. Proteins released from the particles at various NaCl concentrations (up to 2 M) sedimented between 0 and 15 S. 30‐S aggregates corresponding to “informofers” were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCl.

3. Large protein aggregates contaminated the smallest particles (up to 50–60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein‐labelled fractions.

4. About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0–15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross‐linking. A 30‐S protein peak was not detected.

5. The material released from the particles by ribonuclease banded at ϱ= 1.35 g/ml, a higher density than that of free proteins (ϱ= 1.31 g/ml). Treatment with 1 M NaCl after ribonuclease digestion released free proteins, some of which were phosphoproteins (ϱ= 1.31 g/ml) and ribonucleoproteins of higher density. It is suggested that these complexes contain ribonuclease‐resistant RNA.

These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30‐S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins.
Transcription of Supercoiled Mitochondrial DNA by Bacterial RNA Polymerase
Authors: SCHMITT Henri , BECKMANN Jacques S. , LITTAUER Uriel Z.
Published: March 2005

Mitochondrial DNA isolated from cysts of Artemia salina (brine shrimp) was found to have a closed‐circular structure 5.1 μm long and a buoyant density of 1.697 g/cm3. Escherichia coli RNA polymerase can transcribe this DNA. RNA synthesis is greatly stimulated by the σ subunit and is influenced by the presence of KCl. After formation of an initial complex between the enzyme and the mitochondrial DNA only a limited synthesis of RNA took place in the presence of rifampicin.

The products obtained with holo enzyme and core enzyme in the presence of excess nucleoside triphosphates have a mean molecular weight of 1.5 × 106 and greater than 3 × 106 respectively, as determined by acrylamide‐agarose gel electrophoresis. Addition of the termination factor ϱ to the reaction mixture reduced the molecular weight of the products to 1.0 × 106 at a KCl concentration of 0.05 M but not at 0.1 M KCl.

Under limiting UTP concentrations, a few discrete RNA bands of shorter size (about 8 × 105, 105 and 3 × 104 mol. wt) are synthesized. Addition of ϱ factor to the reaction mixture caused the disappearance of the higher‐molecular‐weight band (8 × 105) while the other RNA bands remained.

Little self‐annealing was observed upon heating the [3H]RNA synthesized in vitro at 70 °C. On the other hand, when the [3H]RNA was heated in the presence of unlabeled mitochondrial rRNA, about 54% of the labeled product was resistant to degradation by a mixture of T1 and pancreatic RNase. In hybridization‐competition experiments, annealing of the [3H]RNA synthesized in vitro was reduced by 65% upon addition of unlabeled mitochondrial rRNA. RNA‐RNA annealing is thought to be the main cause for this competition reaction since similar results were obtained by simultaneous or step‐wise hybridization procedures. It is concluded that transcription in vitro of closed circular‐mitochondrial DNA from Artemia by E. coli RNA polymerase proceeds mainly on the anti‐rRNA strand.
Carbamylation of Brain Proteins with Cyanate in vitro and in vivo
Authors: FANDO Juan , GRISOLIA Santiago
Published: March 2005

Unequivocal demonstration of the incorporation of [14C]cyanate into brain proteins as a carbamyl derivative rather than via decomposition to CO2 is presented; this is based on the isolation of radioactive hydantoins and on the loss of radioactivity on hydrolysis of the hydantoins. The influence of conditions on the extent of carbamylation of brain proteins in vitro by cyanate are presented. The optimum pH is about 7 and the carbamylation is dependent on temperature, concentration and time. There was no detectable difference in the rate and/or extent of cyanate incorporation in vitro between normal and previously carbamylated animals. Experiments in vivo show a linear increase in the carbamylation of brain proteins with intraperitoneal injection of cyanate up to the highest concentration tested (2 μmol/g). The extent of carbamylation increases progressively with repeated injections for about 3 days. After this time with further injections there is increased incorporation, but at a lower rate. Experiments in vivo show that animals who have been previously carbamylated with non‐radioactive cyanate incorporate about 25% less cyanate than control animals when given a pulse of [14C]cyanate. Fractionation of the major cell components revealed cyanate incorporation into all fractions but a higher incorporation in the supernatant fraction. When brain was fractionated by differential extraction there was incorporation in all the fractions, including the water, salt and solvent‐insoluble residue. Although some differences were detected, additional studies are needed to determine whether carbamylation affects protein turnover. The higher sensitivity of experiments in vivo for discrimination of protein carbamylation is discussed in relation to the present work and to recent work from others.
Phosphorylation of Vinblastine‐Isolated Microtubules from Chick‐Embryonic Muscles
Authors: PIRAS Marta M. , PIRAS Romano
Published: March 2005

Microtubule protein isolated from muscles of 11‐day‐old chick embryos migrates in gel electrophoresis with sodium dodecylsulphate–5% polyacrylamide as two closely associated bands of molecular weight 55000, which account for more than 90% total protein. Incubation of the micro‐tubular protein with [γ‐32P]ATP and Mg2+ results in a rapid incorporation of radioactivity into the 5% trichloroacetic acid precipitable fraction, without addition of any exogenous protein phosphokinase. The radioactivity can be solubilized by proteolytic enzymes and by alkali, and it migrates together with the faster moving band on gel electrophoresis. Elution of the tubulin band followed by acid hydrolysis gives rise to [32P]phosphorylserine. The protein phosphokinase activity associated to the microtubules is not stimulated by cyclic AMP or cyclic GMP and it can act on exogenous substrates, like casein, histone and phosphovitin. It emerges upon nitration through Bio‐gel P‐300 at a position of molecular weight 54000, separated from the bulk of the 32P radioactivity, which coincides with [3H]colchicine binding (molecular weight 120000). Phosphorylation by ATP (Km= 8 μM) requires Mg2+ or Mn2+. GTP can replace ATP in the phosphorylation reaction. Casein phosphorylation by ATP or GTP is affected only slightly by NaCl and vinblastine, but that of tubulin is affected to different extents, depending on the nucleoside triphosphate used.

The micro tubular protein isolated from: (a) HeLa cells cultured in the presence of 32Pi; (b) muscle and brain slices of chick embryos incubated with 32Pi, and (c) muscles and brains of chick embryos injected with 32Pi, is labelled in all instances with 32P. The radioactivity migrates on gel electrophoresis like tubulin and gives rise upon elution and acid hydrolysis to [32P]phosphoserine. The results in vitro as well as those obtained in intact cells are discussed in connection with the possible role of phosphorylation as a control mechanism of microtubular function.
Transfer of Phosphatidylcholine between Liposomes
Authors: HELLINGS Jan A. , KAMP Hein H. , WIRTZ Karel W. A. , VAN DEENEN Laurens L. M.
Published: March 2005

Liposomes of sufficiently different surface charge can be separated on a DEAE‐cellulose column. This made possible the present study on the transfer of phosphatidylcholine between liposomes consisting of phosphatidylcholine and various amounts of phosphatidic acid. It was demonstrated that the redistribution of [14C]phosphatidylcholine between liposomes as catalyzed by the phosphatidylcholine exchange protein, followed the kinetics of an isotope‐exchange reaction. Furthermore, it was shown that the phospholipid composition of the liposomes affected the activity of the exchange protein.
Phosphorylation of Nuclear Proteins from Bovine Anterior Pituitary Gland Induced by Adenosine 3′:5′‐monophosphate
Authors: JOLICOEUR Paul , LABRIE Fernand
Published: March 2005

In the presence of exogenous protein kinase dependent on adenosine 3′:5′‐monophosphate (cyclic AMP), cyclic AMP leads to an important stimulation of 32P incorporation into chromatin purified from bovine anterior pituitary tissue. Over 80% of 32P incorporated into isolated adenohypophyseal histones is associated with F2b histone while small uptake is measured in F1 and F3 (and its dimer form) histones. Cyclic AMP leads to a 3 to 4‐fold stimulation of incorporation of radioactivity into histone F2b. The radioactivity associated with F2b histone is almost exclusively recovered into two phosphopeptides. Eigthteen bands can be resolved after separation of adenohypophyseal acidic nuclear proteins on 5% sodium dodecylsulfate polyacrylamide gels and their apparent molecular weights range from 12000 to 300000. Protein kinase catalyses the cyclic‐AMP‐dependent phosphorylation of a large number of acidic proteins although preferential labeling occurs in five bands. The present data suggest a possible role of cyclic AMP on the transcriptional activity of the anterior pituitary gland through phosphorylation of histones and non‐histone acidic nuclear proteins.
Transfer of Deuterium from [1‐2H2]Ethanol to Krebs Cycle and Related Acids of Rat Liver in vivo
Authors: CRONHOLM Tomas , MATERN Heidrun , MATERN Siegfried , SJÖVALL Jan
Published: March 2005

The utilization in intermediary metabolism of hydrogens derived from C‐1 in ethanol was studied with the aid of [1‐2H2]ethanol. A gas chromatographic‐mass spectrometric method was developed and used to measure the deuterium content in individual positions of lactate, malate, 3‐hydroxybutyrate, citrate, succinate and fumarate isolated from freeze‐clamped livers of rats metabolizing [1‐2H2]ethanol (95 atoms%2H excess) at a maximal rate.

A steady state of 2H‐labelling was reached within a few minutes. 3‐Hydroxybutyrate contained only 1–2 atoms% of deuterium which was interpreted to indicate that intramitochondrial NADH was essentially unlabelled due to exchange with protons in the NADH dehydrogenase reaction. A low labelling of lactate, 17 atoms%, indicated that the exchange of hydrogens in cytosolic NADH with unlabelled substrates and water was 4 times higher than the rate of ethanol oxidation. Since intramitochondrial oxidation of NADH is not rate limiting, the presence of labelled NADH in the cytosol may indicate that transfer of hydrogen through the mitochondrial membrane is a rate‐limiting step.

As expected from the reversible reactions catalyzed by fumarate hydratase and malate dehydrogenase the deuterium excess at C‐2 and C‐3 of malate and fumarate were about the same, 20 atoms% Citrate had a deuterium excess of about 9 atoms%, assuming that only one hydrogen was labelled. Succinate contained very little deuterium. The labelling of citrate may thus be due either to incorporation of oxaloacetate labelled at C‐3 or to labelling of the same hydrogen in a reaction catalyzed by isocitrate dehydrogenase.

The results indicate that both malate and isocitrate are potential sources of labelled NADPH during oxidation of [1‐2H2]ethanol, and that these two routes are not completely equilibrated under the experimental conditions.
Studies on Seed‐Oil Triglycerides Factors Controlling the Biosynthesis of Fatty Acids and Acyl Lipids in Subcellular Organelles of Maturing Crambe abyssinica Seeds
Authors: APPLEBY Robert S. , GURR Michael I. , NICHOLS Bryan W.
Published: March 2005

The acetyl‐CoA carboxylase activity of all Crambe abyssinica subcellular fractions is very low as indicated by poor conversion of acetate and acetyl‐CoA and a requirement for malonyl‐CoA as the substrate for fatty acid synthesis.

Erucic acid (C22:1) biosynthesis is most active in the oil body fraction, quite active in the denser nuclear/plastid and mitochondrial fractions and absent from the microsomal and cytoplasmic fractions. Erucic acid is formed by direct elongation of oleic acid, not by synthesis from acetate de novo.

Desaturation of added stearyl‐CoA was not demonstrated.

Incorporation of 14C‐labelled acyl groups from fatty acyl‐CoA thioesters into phospholipids occurred in all subcellular fractions except the supernatant from centrifugation at 105000 ×g.

Triglyceride synthesis from 14C‐labelled stearyl‐CoA occurred in all subcellular fractions except the supernatant fraction from centrifugation at 105000 ×g.

Incubations of halved seeds with labelled precursors, followed by subcellular fractionation, supported these findings except that the intact seeds had functional acid‐CoA ligase and acetyl‐CoA carboxylase.

Incubation of halved seeds in the presence of trichloroacetic acid (2.5 mM and 12.5 mM) resulted in substantial inhibition of the biosynthesis of erucic acid but not of other fatty acids. Higher levels of trichloroacetic acid resulted in general inhibition of fatty acid and acyl lipid synthesis.
The Preparation and Characterization of Two Forms of Bovine Galactosyl Transferase
Authors: POWELL Janet T. , BREW Keith
Published: March 2005

Two molecular forms of bovine galactosyl transferase have been purified by affinity chromatography. A unique form, molecular weight 51000, is obtained from bovine colostrum. A lower molecular weight form, 41000, has been obtained by tryptic digestion of the colostral enzyme, or of the three component enzymes isolated from bovine milk. Both forms are active for N‐acetyllactosamine synthase and lactose synthase. The physical characteristics, amino acid composition and steady‐state kinetics of the two forms are compared. Tryptic digestion converts the galactosyl transferase to a more symmetrical form, and alters the nature of the binding of UDP‐galactose to the enzyme · Mn2+ complex.
Modification of Phenylalanyl‐tRNA Synthetase from Escherichia coli by Histidine‐Specific Reagents
Authors: HENNECKE Hauke , BÖCK August
Published: March 2005

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.

All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.

Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.

During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.
Studies on the Specificity of Action of Bacteriophage T4 Lysozyme
Authors: MIRELMAN David , KLEPPE Gunnar , JENSEN Harald B.
Published: March 2005

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endo‐acetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide side‐chains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted.
Study of Coenzyme Binding Site of Octopine Dehydrogenase Using Analogues of NAD+
Authors: OLOMUCKI Anna , THOMÉ‐BEAU Françoise , BIELLMANN Jean‐François , BRANLANT Guy
Published: March 2005

The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring.

The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect.

Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide–8‐bromoadenine dinucleotide, nicotinamide–8‐thioadenine dinucleotide and nicotinamide 1:N6‐ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes.

Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding.

The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary but not ternary complexes.

The case of 1,4,5,6‐tetrahydronicotinamide–adenine dinucleotide which does not give ternary complexes like NADH is discussed.

The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described.

The protective effects of coenzyme analogues towards essential–SH group were also studied.
The Self‐Association of Chicken‐Erythrocyte Histones
Authors: DIGGLE John H. , McVITTIE John D. , PEACOCKE Arthur R.
Published: March 2005

The self‐association of the separate histone fractions isolated from chicken erythrocytes has been studied in solution at a number of different pH values and ionic strengths. The apparent molecular weights of the histones were determined over a range of macromolecular concentrations using the techniques of osmotic pressure and sedimentation equilibrium. Histone F2c (H5) did not associate under any of the conditions investigated whereas the other histone fractions all appeared to undergo self‐association forming dimers, dimers of dimers, etc. The degree of association increased with the pH and ionic strength of the medium. The tendency to aggregate increased in the order; histone F2c (H5) (non‐aggregating), histone F2b (H2B), histone F2a2 (H2A), histone F3 (H3), histone F2a1 (H4) (highly aggregating).

In the case of histone F2a2 (H2A) at pH 3.0 and ionic strength 0.1, the apparent weight‐average molecular weight was determined at a number of macromolecular concentrations at five different temperatures. The self‐association was analysed according to the method of Adams (published by Beckman Instruments Inc. in 1967) and shown to be a monomer‐dimer‐tetramer equilibrium. The association constants were evaluated at each of the temperatures studied and from their variation with temperature the values of the enthalpy and entropy of association were calculated. The intermolecular association was characterised by only a small change in enthalpy but a large, positive, change in entropy. This suggests that the association of histones at acid pH is due to hydrophobic interactions between the relatively uncharged segments of like polypeptide chains.
Initiation Specificity of the Poly(cytidylic acid)‐Dependent Qβ Replicase Activity
Authors: FEIX Günter , SANO Hiroshi
Published: March 2005

The initiation specificity of RNA synthesis catalysed by the poly(C)‐dependent Qβ replicase activity was investigated with various synthetic ribopolymers as template. It was found that the initiation efficiency of a series of oligo(C) with various chain lengths is proportional to the template size. Synthetic riboheteropolymers containing cytidylic acid were accepted as templates only if they contained at their 3′ end a cytidylic acid sequence of more than 5 nucleotides. Such an oligocytidylate sequence served also as an initiator sequence for copying the non‐cytidylic‐acid‐containing part of the heterotemplate. RNA synthesis always began with the incorporation of GTP, even if the 3′‐terminating nucleotide of the template was not cytidylic acid.
Interactions of Colipase with Bile Salt Micelles
Authors: SARI Hélène , ENTRESSANGLES Bernard , DESNUELLE Pierre
Published: March 2005

The finding reported in the preceding paper that colipase is able to bind one sodium taurodeoxycholate micelle per molecule was confirmed by dialysis and spectrophotometry. Dialysis in the presence of labelled sodium taurodeoxycholate provided a direct qualitative proof of taurodeoxycholate binding to colipase. This binding was found to occur only above the critical micelle concentration. But, dialysis did not give any information about the composition of the associations, because equilibrium was not attained at the end of the assays.

Addition of sodium taurodeoxycholate above the critical micelle concentration was also observed to induce a strong perturbation of the ultraviolet spectrum of one or several of the three tyrosines of colipase. The variation of the perturbation as a function of sodium taurodeoxycholate concentration was consistent with the binding of a single micelle to colipase. The dissociation constant calculated in “micelle molarity” was approximately 1 × 10‐4 M.

The colipase‐bile salt micelle association can fix one molecule of lipase to form a ternary complex which represents an interesting model of a protein‐protein interaction mediated by an organized lipid structure. The ternary complex is probably also a model for lipase‐substrate interactions in the presence of an amphipath.
The Isolation of Two Different Lipopolysaccharide Fractions from Various Proteus mirabilis Strains
Authors: GMEINER Jobst
Published: March 2005

Four distinct Proteus mirabilis strains were extracted by the phenol/water procedure. After ultra‐centrifugation of the dialyzed water phase, the pelleted lipopolysaccharide was purified and analyzed. The sugar composition of this lipopolysaccharide fraction I was similar for all four strains, containing only small amounts of strain‐specific constituents. A second lipopolysaccharide fraction was isolated from the supernatant above (termed L1 fraction) after removal of nucleic acids. DEAE‐cellulose chromatography indicated that this material is not a polysaccharide but rather a water‐soluble lipo‐polysaccharide containing strain‐specific constituents such as uronic acids, amino acids, amino sugars, neutral sugars, ethanolamine and phosphate, depending on the strain from which lipo‐polysaccharide II was isolated.
Molecular Morphology of Ribosomes
Authors: MICHALSKI Chester J. , SELLS Bruce H.
Published: March 2005

Using either soluble or solid‐state lactoperoxidase, a comparison was made between the enzymic iodination of ribosomal proteins iodinated as 30‐S and 50‐S subunits or as 70‐S monosomes. Proteins S7, S11 and S12 of the 30‐S subunit and proteins L2, L11, L26 and L28 of the 50‐S subunit were labelled to a greater extent in isolated particles than in the 70‐S ribosome. In contrast, proteins S4, S6, S19 and S20 were labelled to a lesser extent in the isolated subunit. No significant differences were observed in the iodination patterns of ribosomes iodinated in the presence of soluble lactoperoxidase and those iodinated in the presence of lactoperoxidase bound to Sepharose 4B. It is suggested that the 30‐S subunit undergoes a conformational change during its association with the 50‐S subunit to form a 70‐S monosome. Implications from results obtained with solid‐state lactoperoxidase‐catalyzed iodination of ribosomal proteins are also discussed.
RNA‐Protein Interactions in the Ribosome
Authors: SPIERER Pierre , ZIMMERMANN Robert A. , MACKIE George A.
Published: March 2005

Limited digestion of the Escherichia coli 50‐S subunit permits the isolation of two large fragments of the 23‐S RNA. One arises from the 5′ end of the 23‐S RNA and contains about 1200 nucleotides. The other arises from the 3′ end of the 23‐S RNA and contains about 2000 nucleotides. Each of the ten 503 proteins known to bind specifically and independently to the 23‐S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5′ terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3′ terminal segment contains the specific binding sites for proteins L1, L2, L3, L6, L13, L16 and L23.
Kinetic Equivalence of the Active Sites of Alcohol Dehydrogenase from Horse Liver
Authors: HADORN Matthias , JOHN Vivian A. , MEIER Felix K. , DUTLER Hans
Published: March 2005

The reduction, catalysed by liver alcohol dehydrogenase, of benzaldehyde in the presence and absence of pyrazole, and the oxidation of benzyl alcohol and cyclohexanol in the presence of isobutyramide, has been measured by the stopped‐flow technique. In performing these experiments particular care was taken to purify the enzyme, coenzymes, substrates and inhibitors, and to minimise as much as possible the effects of a blank substrate reaction. The calculation of the amount of substrate converted to product during the various phases of the transient process was based on the absorption coefficients for the enzyme‐coenzyme and enzyme‐coenzyme‐inhibitor complexes determined in the absence of substrate. The results show that the two active sites of liver alcohol dehydrogenase are kinetically equivalent and that the enzyme does not exhibit half‐of‐the‐sites reactivity.
Purification and Characterization of a New Factor which Restores Protein Synthesis in a Conditionally Lethal Mutant of Escherichia coli
Authors: VAN DER MEER John P. , GANOZA M. Clelia
Published: March 2005

The mutant Escherichia coli strain, N4316, has a temperature‐sensitive defect in a protein factor required for translation in vitro of bacteriophage f2 RNA. We have purified the normal counterpart of this factor from a wild‐type strain, using as an assay its ability to restore the activity of mutant extracts at non‐permissive temperature. Our final preparation is free of known initiation, propagation, and release factors, proving that the factor is a new component required for translation.

The new factor has a molecular weight of 95000 with preliminary data suggesting a subunit structure. 70% of this protein is found in the soluble‐cell fraction, the rest being associated with 70‐S ribosomes. Kinetic analyses indicate that the factor acts early in translation.

Expression of the defect is highly dependant on the Mg2+ concentration, no temperature‐sensitivity being apparent at 15 mM Mg2+. At lower Mg2+ concentrations, the defect is expressed only with natural mRNAs such as f2 RNA, and not with artifical polymers such as poly(U). This specificity suggests that the factor may function in events coded by special sequences in the natural messengers.
Conformational Changes of Alamethicin Induced by Solvent and Temperature
Authors: JUNG Günther , DUBISCHAR Norbert , LEIBFRITZ Dieter
Published: March 2005

13C nuclear magnetic resonance (NMR) and circular dichroism (CD) have been used for studies on the conformation of alamethicin. The 13C NMR spectrum is assigned with the aid of signals of synthetic partial sequences and selective proton decoupling. The solvent and temperature‐dependence of the 13C NMR spectra, T1 measurements and the use of lanthanide‐shift reagents allow the differentiation between the amino acids belonging to a rigid α‐helical portion of the alamethicin sequence and those belonging to a more flexible part. The 13C NMR results are in agreement with results obtained from extended solvent and temperature‐dependent CD studies which indicate a highly stabilized nonpolar and intrachenar α‐helical part. The concentration‐dependence of the CD spectrum of alamethicin in a nematic phase revealed aggregation phenomena which might simulate those observed in natural and synthetic membranes. After dissolving alamethicin in aqueous alcohol there is a time‐dependence of the ellipticity of the Cotton effects showing a sort of memory effect on the mode of dissolution. Four different conformations can be characterized by CD spectra depending on the solvent and concentration. A model illustrating the dynamic conformations and aggregation phenomena within a membrane is proposed.
Evidence for an Essential Lysine in Glucose‐6‐Phosphate Dehydrogenase from Leuconostoc mesenteroides
Authors: MILHAUSEN Michael , LEVY H. Richard
Published: March 2005

Pyridoxal 5′‐phosphate inhibits glucose‐6‐phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki= 0.04–0.06 mM.

This inhibition is competitive with respect to glucose 6‐phosphate and non‐competitive with respect to NADP+ or NAD +. Interaction between enzyme and excess pyridoxal 5′‐phosphate follows pseudo‐first‐order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme.

Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5′‐phosphate. Dissociation constants for NADP+ and glucose 6‐phosphate were determined from their effects on the kinetics of enzyme–inhibitor interaction.

Reaction of the enzyme with pyridoxal 5′‐phosphate produces a typical Schiff‐base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine.

The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N6‐pyridoxyllysine can be identified by paper chromatography.

The enzyme is inhibited irreversibly by 1‐fluoro‐2,4‐dinitrobenzene, even in the presence of excess 2‐mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ and glucose 6‐phosphate protect against inhibition by 1‐fluoro‐2,4‐dinitrobenzene. The absorption spectrum of dinitrophenyl‐enzyme corresponds to that for dinitrophenylated amino groups.

These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6‐phosphate.

It is proposed that a group of “active lysine” proteins may exist (in analogy with the “active serine” enzymes), which share a common structural feature at their substrate‐binding site and to which pyridoxal 5′‐phosphate binds specifically.
Studies of Intracellular Thymidine Nucleotides
Authors: OHKAWA Tsutomu
Published: March 2005

In a thymine‐deprived culture, the mutant cells (deficient in dTDP‐glucose pyrophosphorylase activity and named Ter‐15) lose viability at a faster rate, form longer filaments for the first 60 min and lose thymidine nucleotides and dTDP‐sugar pools at a faster rate for the first 15 min than those of the parent cells, but the dTDP‐sugar pool in the parent cells is maintained at high concentration for the first 90 min during thymine starvation.

In the recovery of cell growth after re‐addition of thymine into the thymine‐deprived culture, parent cells recommence growth immediately, but the mutant cells (Ter‐15) show a lag‐phase for 45 min after which time their growth recommences.

The rate of dTTP synthesis for the first 10 to 15 min after re‐addition of thymine to thymine‐deprived cultures of parent and mutant (Ter‐15) cells is three‐fold higher than that of a thymine nondeprived culture (control), but the rates of dTMP and dTDP‐sugar syntheses are the same as those of the control.

The total DNA synthesis after re‐addition of thymine is equal to that of the control, and the period of thymine starvation other than the number of viable cells during thymine starvation plays an important role. After separation of the filament cells from normal‐sized cells by sucrose gradient centrifugation, the initial rate of DNA synthesis of filament cells is three‐fold faster than that of normalsized cells.

These results show that the dependency of DNA synthesis upon dTTP concentration is maintained after re‐addition of thymine into thymine‐deprived culture.
Nuclear Protein Kinases from Murine Cells
Authors: SCHLEPPER Jutta , KNIPPERS Rolf
Published: March 2005

Three protein kinase activities are found in nuclei from three different murine cells (Ehrlich ascites cells, mouse L cells and rat glioma cells). Two of these activities are soluble, one is bound to chromatin. The soluble enzymes are similar, if not identical, to the cytoplasmic protein kinases. The chromatinbound, adenosine‐cyclic‐3′: 5′‐monophosphate‐independent enzyme is not found in the cytoplasm. This enzyme is composed of one subunit with a molecular weight of 80000–90000. Some biochemical properties of this enzyme are described. A brief description of a nuclear enzyme, which dephosphorylates phosphorylated histones, is also given
Investigations of the Structure of 3‐Methylcrotonyl‐CoA Carboxylase from Achromobacter
Authors: SCHIELE Ulrich , NIEDERMEIER Rosa , STÜRZER Magdalena , LYNEN Feodor
Published: March 2005

It was shown by gel electrophoresis in sodium dodecylsulphate solution that 3‐methylcrotonylCoA carboxylase from Achromobacter IVS is composed of two different subunits with molecular weights of about 78000 and 96000, respectively. The biotin is bound to the heavier subunit. It was previously found that 3‐methylcrotonyl‐CoA carboxylase contains four biotin molecules per complex. A complex composed of four of each subunit would thus have a molecular weight of about 700000. This is compatible with the molecular weight of 760000 determined earlier by analytical ultracentrifugation.

Both subunits were isolated preparatively. As the subunits, unlike the complex, are very sensitive to oxygen, special precautions had to be taken during isolation. The biotin‐containing subunit was isolated by chromatography on DEAE‐cellulose in 5 M urea. It no longer catalyzed the overall reaction, yet could still carboxylate free biotin. The biotin‐free subunit was separated after dissociation of the enzyme by three‐days' dialysis at pH 9.8 under nitrogen. On chromatography over a Sepharose‐bound avidin column, the biotin‐subunit was fixed and the biotin‐free subunit was eluted unretarded. The latter subunit showed no enzymic activity. After the addition of the biotin‐containing subunit, overall activity was regenerated.

The speed of reassociation is very much enhanced by 3‐methylcrotonyl‐CoA. It was shown by reassociation experiments under different conditions that probably an initial complex AxBy is formed, possessing a binding site for 3‐methylcrotonyl‐CoA. Upon, the binding of this substrate the conformation may be changed to a form favourable for reconstitution.

Finally, the structures of biotin enzymes from different sources are compared. In the course of evolution there is a tendency toward integration of the different constituent proteins into only one polypeptide chain.
Biochemical Research on Oogenesis
Authors: WEGNEZ Maurice , MAZABRAUD André , DENIS Herman , PÉTRISSANT Guy , BOISNARD Monique
Published: March 2005

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs.

Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.
Purification and Properties of a Periplasmic Aminoendopeptidase from Escherichia coli
Authors: LAZDUNSKI Claude , BUSUTTIL Jeanine , LAZDUNSKI Andrée
Published: March 2005

A periplasmic aminoendopeptidase from Escherichia coli has been purified to homogeneity. It is a monomer of molecular weight 45000 and containing one – SH group that is necessary for catalytic activity. The study of its substrate specificity indicated that the enzyme has both aminopeptidase and endopeptidase activity. The pH optimum for l‐alanine p‐nitroanilide hydrolysis is between 7 and 7.5 and that for 125I‐labelled casein proteolysis between 7.3 and 7.6. The activation energy for the hydrolysis of l‐alanine p‐nitroanilide was calculated to be 5.3 kcal × mol−1 (22.2 kJ × mol−1).
Subcellular Distribution of Histone‐Degrading Enzyme Activities from Rat Liver
Authors: HEINRICH Peter C. , RAYDT Gerhard , PUSCHENDORF Bernd , JUSIĆ Mirna
Published: March 2005

Chromatin prepared from liver tissue contains a histone‐degrading enzyme activity with a pH optimum of 7.5–8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone‐degrading enzyme activity was assayed with radioactively labelled total histories from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone‐degrading enzymes. A pH optimum around 4.0–5.0 was found for the lysosomal fraction, whereas 7.5–8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase‐free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria.

The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin‐associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes.

The chromatin‐associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikreín‐trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin‐associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone‐degrading enzyme activity.
Inactivation of Bacillus cereusβ‐Lactamase by Halogenated Isoxazolylpenicillins
Authors: STROM Roberto , RAVAGNAN Giampiero , SALFI Vincenzo
Published: March 2005

Upon preincubation with halogenated isoxazolyl‐penicillins, the activity of Bacillus cereusβ‐lactamase is decreased by a factor proportional to the inhibitor concentration. This inactivation, which concerns essentially the catalytic rate constant of the enzyme, occurs only at or above 37°C with cloxacillin and dicloxacillin, and even at 25°C with flucloxacillin. The dependence of the inactivation rate upon inhibitor concentration is consistent, in the former case, with the occurrence of a bimolecular reaction, and in the latter case, with a bimolecular process followed by a monomolecular step.
Pyruvate Carboxylase Affinity Labelling of the Magnesium Adenosine Triphosphate Binding Site
Authors: EASTERBROOK‐SMITH Simon B. , WALLACE John C. , KEECH D. Bruce
Published: March 2005

The 2′,3′‐dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg‐oATP2−) was shown to be a linear competitive inhibitor with respect to MgATP2− in both the acetyl‐CoA‐dependent and ‐independent activities of the enzyme but was a non‐competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg‐oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme. Although bicarbonate, pyruvate and oxaloacetate were ineffective, both MgATP2− and acetyl‐CoA protected the enzyme against this chemical modification. At 100% inactivation, 1.1 ± 0.1 mol of Mg‐oATP2− were bound to the enzyme per mole of biotin. Acetyl‐CoA had no effect on this stoichiometry.

Chromatography of samples of an enzymic digest of Mg‐o[14C]ATP2− ‐labelled enzyme revealed one major band of radioactivity which co‐chromatographed with authentic Lys‐oATP.
Elongation Factor T from Bacillus stearothermophilus and Escherichia coli
Authors: WITTINGHOFER Alfred , LEBERMAN Reuben
Published: March 2005

Homogeneous preparations of elongation factors EF‐Tu and EF‐Ts from Bacillus stearothermophilus have been obtained with specific activities of 20000 ± 2000 and 500000 ± 50 000 units/ mg, respectively. By dodecylsulphate‐polyacrylamide gel electrophoresis the molecular weight of EF‐Tu was found to be 49000 ± 2000 and of EF‐Ts 35500 ± 1000. Nucleotide‐free EF‐Tu was prepared by using ITP as a GDP‐binding‐site‐directed analogue. EF‐Tu was shown to contain two sulphydryl groups, one reacting fast and one slowly with N‐ethylmaleimide and 5,5′‐dithio‐bis(2nitrobenzoic acid) under non‐denaturing conditions. The same reagents were shown to react with the three sulphydryl groups of EF‐Ts in the native state. The heat stabilities of EF‐Tu and EF‐Ts are reversed with respect to the Escherichia coli factors, EF‐Tu being the more stable protein; even nucleotide‐free EF‐Tu is relatively stable with a half‐life at room temperature of about 35 h.
A Calorimetric Study of the CO Bohr Effect of Monomeric Haemoglobins
Authors: GERSONDE Klaus , NOLL Leo , GAUD Henry T. , GILL Stanley J.
Published: March 2005

A calorimetric study has been made of the heats of CO reaction with the monomeric haemoglobins of Chironomus thummi thummi III and IV as a function of pH. The number of Bohr protons released at pH 7.1 was determined from heats of reaction in different buffers as 0.19 and 0.31 mot H+/mol CO for haemoglobin III and IV respectively. The heat of the Bohr ionization process was found to be 6 and 8 kcal/mol H+ (25 and 34 kJ/mol) for the haemoglobins III and IV. These values are consistent with values found for histidine groups. A pH‐independent part of the reaction enthalpy was determined as ‐ 19.7 kcal/mol CO (‐ 82.4 kJ/mol). The same reaction with myoglobin is less exothermic. From the combination of ΔG° and ΔH° values TΔG° values have been calculated. It was found for both haemoglobins that the entropy of reaction is greater by 2 cal K‐1 mol‐1 (8.4 J K‐1 mol ‐1) at pH 9.5 as compared to pH 6.0.