bluuebunny/crossref_metadata_2025 huggingface dataset:
The Interaction of Daunomycin with Polydeoxynucleotides
Authors: PHILLIPS Don R. , DiMARCO Aurelio , ZUNINO Franco
Published: March 2005

The ability of daunomycin to bind to various DNA polymers has been studied by thermal denaturation, spectrophotometric analysis and inhibition of the polymerisation reactions catalysed by Escherichia coli DNA polymerase I and rat liver DNA polymerase α. The quantitative binding measurements revealed that the antibiotic binds tightly to all synthetic polydeoxynucleotides studied. The results demonstrated that daunomycin can bind with equal affinity to dG · dC or dA · dT base‐paired sequences. However, the number of binding sites per nucleotide for poly(dA) · poly(dT) is significantly lower than that found for poly(dA‐dT) · poly(dA‐dT), thus indicating an appreciable preference of the drug for the alternating copolymer.

The inactivation of the template properties of the synthetic DNA polymers in the DNA polymerase system is consistent with their daunomycin binding ability. However, a lack of correlation was observed between the drug binding ability of different DNA polymers and the binding‐induced stabilisation of the double helix to heat denaturation.
The Steady‐State Kinetics of Yeast Phosphoglycerate Kinase
Authors: SCOPES Robert K.
Published: March 2005

A re‐investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3‐bis‐phosphoglycerate formation has been carried out, covering a 1000‐fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used.

Kinetic plots proved to be non‐linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation.

The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration.

The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons.

Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5‐fold with 0.2 mM MgATP and 0.2 mM 3‐phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects.

The presence of 20 mM or more Na2SO4 converted non‐linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor.

Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.
X‐Ray and Neutron Small‐Angle Scattering Studies of the Complex between Protein S1 and the 30‐S Ribosomal Subunit
Authors: LAUGHREA Michael , ENGELMAN Donald M. , MOORE Peter B.
Published: March 2005

X‐ray and neutron solution scattering experiments have been done to investigate the influence of the binding of ribosomal protein S1 on the conformation of the 30‐S ribosomal subunit of Escherichia coli. The following conclusions were made.

The alterations (if any) in conformation of the non‐S1 parts of the 30‐S subunit induced by S1 binding are too small to be detected (< 0.1 nm change in radius of gyration).

The center of gravity of protein S1 bound to the 30‐S subunit is quite far from the center of gravity of the particle (≅ 7.5 nm).
The Redox Potential of Dithionite and SO−2 from Equilibrium Reactions with Flavodoxins, Methyl Viologen and Hydrogen plus Hydrogenase
Authors: MAYHEW Stephen G.
Published: March 2005

It has been shown that redox equilibria can be formed between dithionite ion (plus SO−2) and (bi)sulphite, and the low‐potential electron carriers flavodoxin and methyl viologen. The equilibria were established either by treating the oxidized electron carriers with dithionite, or by treating flavodoxin hydroquinone or methyl viologen semiquinone with (bi)sulphite. Similar redox equilibria were established between dithionite/(bi)sulphite and hydrogen using catalytic amounts of hydrogenase in the presence of a low‐potential electron carrier. The effects of pH and temperature on the equilibria were determined.

The equilibria were analyzed to determine the redox potential of the dithionite/(bi)sulphite system. In accordance with the results of earlier kinetic studies, it was assumed that the reductant in dithionite solutions is the dissociation product SO−2. The calculated midpoint redox potential E' for the couple SO−2/HSO−3 at pH 7 and 25 °C was –0.66 V. The reductant is present largely as the dimer at concentrations of dithionite above about 10nM. Consequently, the midpoint potential, Em, of dithionite solutions becomes less negative as the concentration of dithionite is increased (ΔEm/Δlog S2O2−4= 29 mV). The theoretical potential of a solution of 1 M S2O2−4 and 2 M (bi)sulphite at pH 7 was calculated to be –0.386 V. This value is 59 mV more negative than that determined in 1911 by potentiometry, but considerably more positive than other values in the literature. The effects of pH on the equilibria showed that E' is controlled by the pK of (bi)sulphite at 6.9; the slope ΔE'/ΔpH was –59 mV below the pK and – 118 mV above the pK. The effects of temperature on the equilibria suggested that Em for dithionite changed by –1.6 mV/°C for a rise in temperature between 2 °C and 40 °C. If sodium dithionite is contaminated with small amounts of (bi)sulphite, its addition in large excess to a low potential electron carrier can cause oxidation of the carrier.
Metabolic Stability of the Extrachromosomal Ribosomal RNA Genes in the Slime Mould Physarum polycephalum
Authors: HALL Len , GUBLER Ueli , BRAUN Richard
Published: March 2005

The rRNA genes of the slime mould Physarum polycephalum are located on free, linear DNA molecules of a discrete size, Mr= 38 × 106. Using an isotope dilution technique we have examined the metabolic stability of these extrachromosomal genes during active, balanced growth. Microplasmodia, prelabelled with [3H]thymidine, were used to prepare synchronous surface plasmodial cultures which were subsequently grown on unlabelled medium. The gross synthesis of ribosomal DNA was then determined over three consecutive mitotic divisions from the ratio of 3H to 14C in a hybrid formed between the extracted ribosomal [3H]DNA and a [14C]rRNA probe. It was found that ribosomal DNA, like chromosomal DNA, is completely stable during active growth.
Investigation of the Interaction of Triethyltin with Rat Liver Mitochondria Using Binding Studies and Mössbauer Spectroscopy
Authors: FARROW Barry G. , DAWSON Alan P.
Published: March 2005

The binding of triethyltin to rat liver mitochondria is measured and the results indicate the presence of a three component system. This involves triethyltin binding to two specific sites, one of high affinity, which is found to have a concentration similar to that found for the F1‐ATPase complex and one of lower affinity. The third component is attributed to partition of triethyltin into the mitochondrial membrane.

Non‐specific partition is shown by inhibition of triethyltin binding to the low affinity site with N‐ethylmaleimide and to the high affinity site by buffer pH values lower than pH 5.7. Combination of these two conditions leaves a Scatchard plot which can be interpreted as being due to triethyltin partitioned into the mitochondrial membrane.

The selective inhibition of triethyltin binding to the low affinity site by N‐ethylmaleimide preincubation indicates the presence of a thiol group in the low affinity site and the absence of N‐ethylmaleimide‐reactive thiol groups necessary for triethyltin binding in the high affinity site.

Mössbauer spectra of triethyltin bound to mitochondria also seem to be derived from the same three component system. N‐Ethylmaleimide preincubation assigns an absorption doublet to low affinity bound triethyltin and the concentration dependence of the absorption intensities assigns a second doublet to partitioned triethyltin.

Washing triethyltin pretreated mitochondria leaves an absorption doublet which is assigned to high affinity bound triethyltin. This assignment is confirmed by binding and ATPase activity studies before and after this washing procedure.

Triethyltin bound to the high and low affinity sites is found, by the ratio of the Mössbauer quadrupole splitting and the isomer shift obtained, to be four coordinate or possibly cis‐five coordinate.

The assignment of the absorption doublet, from triethyltin in the high affinity site environment, to a four coordinated triethyltin‐mitochondrial membrane complex is suggested by the investigation of some intramolecularly coordinated triorganotin compounds.
Study of Follitropin Receptors in Testis Using a Homologous System
Authors: MAGHUIN‐ROGISTER Guy , CLOSSET Jean , COMBARNOUS Yves , HENNEN Georges , DECHENNE Charles , KETELSLEGERS Jean‐Marie
Published: March 2005

The properties of follitropin receptors in immature porcine testis were determined using highly purified porcine follitropin.

The characteristics of follitropin binding to a subcellular fraction rich in plasma membranes were studied using a 125I‐labelled follitropin with high specific activity (75–100 Ci/g) and high binding activity. The binding is dependent on time, temperature and pH.

It is specific to follitropin as demonstrated by the very low binding activity of the follitropin α and β subunits and of the other glycoprotein hormones. Scatchard analysis of binding data indicated an equilibrium association constant of 2 × 1010 M−1 and a concentration of high affinity binding sites of 500 fmol/mg membrane proteins.

A sensitive radio‐ligand receptor assay was developed. Fifty percent inhibition of binding was obtained with as little as 2 ng of porcine follitropin. Ovine and bovine follitropins and pregnant mare serum gonadotropin gave binding inhibition curves parallel to that given by porcine follitropin. With equine and human follitropin, significantly different slopes were recorded.

Kinetics of dissociation of labelled follitropin from its testis receptors showed the presence of at least two compartments with fast and slow dissociation rate constants. The ratio between the sizes of the slow and fast compartments appeared dependent upon preincubation time.

A temporal correlation was observed between binding of follitropin to testis receptors and activation of membrane bound adenylate cyclase.
The Binding of Kirromycin to Elongation Factor Tu
Authors: PINGOUD Alfred , URBANKE Claus , WOLF Heinz , MAASS Guenter
Published: March 2005

The influence of kirromycin on the elongation factor Tu (EF‐Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF‐Tu · GDP and EF‐Tu · GTP was determined by circular dichroism titrations to be 4 × 106 M−1, and to EF‐Tu · GTP · aa‐tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 × 106 M−1.

In the presence of kirromycin the binding of aminoacyl‐tRNAs to EF‐Tu · GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl‐tRNA is more accessible to the non‐enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.
Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane , DAVIDSON Barrie E.
Published: March 2005

The effects of phenylalanine, NaCl and pH on the conformation of chorismate mutase/prephenate dehydratase have been investigated, using measurements of far and near‐ultraviolet circular dichroic spectra and ultraviolet difference spectra. At pH 8.2 in 20 mM Tris‐Cl buffer the enzyme was found to contain 10–20% helix and 40–50%β‐structure. There was little or no change in these values on the addition of 1 mM phenylalanine (the allosteric effector) or 0.4 M NaCl or by decreasing the pH to 7.4.

Both phenylalanine and NaCl caused significant changes in the conformation of the enzyme. The most prominent of these was the movement of a tryptophan residue into a more hydrophobic environment. There was also a slight perturbation of this tryptophan when the pH was decreased to 7.4. The conformational changes can explain sigmoidal kinetic behaviour observed previously [Gething et al. (1976) Eur. J. Biochem. 71, 317–325].
Characterisation of the Binding of Virginiamycin S to Escherichia coli Ribosomes
Authors: de BETHUNE Marie‐Pierre , NIERHAUS Knud H.
Published: March 2005

Virginiamycin S is an inhibitor of protein synthesis in vivo. In this paper we show by equilibrium dialysis that it binds specifically to the 50‐S subunit of Escherichia coli ribosomes, with one binding site per subunit. This binding is not altered by the presence of chloramphenicol, tetracycline or puromycin but is competed for by erythromycin. Using the splitting‐reconstitution method, it could be demonstrated that protein L16 is absolutely required for the binding of virginiamycin S to the 50‐S subunit.
Evidence for Essential Histidine Residues in Tryptophanyl‐tRNA Synthetase
Authors: FAVOROVA Ol'ga O. , MADOYAN Igor A. , KISSELEV Lev L.
Published: March 2005

Beef pancreas tryptophanyl‐tRNA synthetase (Mr 60000 × 2) reacts with diethyl pyrocarbonate at pH 6.0. A differential spectrum of modified versus native enzyme as well as the restoration of the initial spectrum after NH2OH treatment of the modified enzyme prove the formation of carbethoxy histidine residues. In 8 M urea, 22 histidine residues are carbethoxylated by diethyl pyrocarbonate in accord with the total histidine content of the enzyme. In the native enzyme only 4–5 histidine residues react at equal rate whereas the others are resistant to modification. The circular dichroism spectra of the native and modified protein are similar. The modified enzyme does not dissociate into subunits.

After modification of accessible histidine residues the enzymatic activity sharply decreases both in the reaction of ATP‐[32P]pyrophosphate exchange and that of tRNA aminoacylation. The modified enzyme is almost completely reactivated after treatment with NH2OH. The rate of the enzyme inactivation in the aminoacylation reaction approaches quite closely that of carbethoxylation of the histidine residues; a complete loss of the ability of tryptophanyl‐tRNA synthetase to catalyze tryptophanyl‐tRNA synthesis is brought about by the modification of 2–3 histidine residues per dimer. The modified enzyme losses almost completely its ability to bind tryptophanyl‐tRNA or uncharged tRNA. A conclusion is made that the accessible histidine residues are involved directly or indirectly in the tRNA binding. The ATP‐[32P]pyrophosphate exchange is less affected with modification of the enzyme by diethyl pyrocarbonate than the tRNA acylation reaction. All substrates protect the tryptophanyl‐tRNA synthetase against inactivation with diethyl pyrocarbonate; the most effective protectors are tryptophan and tryptophan plus ATP‐Mg2+ for the exchange reaction. Modified peptide(s) was isolated by diagonal electrophoresis.
Electron‐Microscopic Demonstration of Terminal and Internal Initiation Sites for cDNA Synthesis on Vitellogenin mRNA
Authors: WAHLI Walter , WYLER Toni , WEBER Rudolf , RYFFEL Gerhart U.
Published: March 2005

cDNA synthesized on purified vitellogenin mRNA from Xenopus liver was hybridized to the template in formamide/urea at 22°C to avoid degradation of the RNA. The hybrids formed were visualized by spreading for electron microscopy. Contour length measurements proved that most of the RNA molecules in the hybrids were still intact showing the expected molecular weight of 2.3 × 106. The hybridized cDNA corresponded on the average to 12% of the RNA length. In about 80% of the molecules the cDNA was located at one end. Since cDNA synthesis was primed by oligo(dT), the terminal duplex region marks the 3′ end of the vitellogenin mRNA molecule. Internal duplex regions were mainly located at a specific position starting about 2800 nucleotides from the 3′ end. Since the cDNA hybridizing at the internal position could specifically be synthesized on a vitellogenin RNA fragment isolated on poly(U)‐Sepharose as an oligo(A)‐containing RNA, we conclude that cDNA synthesis is not only initiated by the poly(A) of the 3′ end, but also by a specific internal sequence.
On the Activity of RNA Polymerase B in Lysates from Ehrlich Ascites Cells
Authors: DREYER Christine , HAUSEN Peter
Published: March 2005

Transcription by endogenous RNA polymerase B in lysates of Ehrlich ascites cells was investigated. The enzyme exhibits two salt optima at 0.025 M and at 0.3 M (NH4)2SO4 respectively. Preincubation of the cells with the nucleoside analogue 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole results in an inactivation of the polymerase molecules active under condition of low salt. This indicates two functional states of the enzyme in vivo.

Initiations of RNA chains by polymerase B do not occur in vitro as judged by the incorporation of [β‐32P]GTP. Thus the two functional states seem to be both elongating polymerase molecules.

Polymerase B does not occur in the lysates in a state ready to initiate on an exogenous template, in contrast to polymerase A and C which do occur in free form. Pretreatment with dichlororibofuranosylbenzimidazole in vivo does not result in an accumulation of free polymerase B.
Effect of Cycloheximide on Maturation of Replicative Intermediates into High‐Molecular‐Weight DNA in Tetrahymena
Authors: WESTERGAARD Ole , MARCKER Kjeld A. , LEER Johan Chr.
Published: March 2005

Replicative intermediates of discrete size (∼ 41 S) are observed in the eukaryotic organism Tetrahymena pyriformis, when the organism is grown under defined physiological conditions. The intermediates (believed to represent replicons) are synthesized and accumulated over longer periods of time (< 90 min), if the cells are treated with low concentrations of cycloheximide. Under these conditions the rate of total DNA synthesis is only slightly inhibited (< 15%), while maturation of intermediate DNA into high‐molecular‐weight DNA is completely blocked (> 98%). Cycloheximide appears to inhibit the maturation process more specifically than other protein synthesis inhibitors.

Studies of the accumulated intermediates on alkaline buoyant density gradients demonstrate that initiation of new putative replicons occurs during treatment with cycloheximide.
Specific Ribonucleases Involved in Processing of tRNA Precursors of Escherichia coli
Authors: SHIMURA Yoshiro , SAKANO Hitoshi , NAGAWA Fumikiyo
Published: March 2005

Ribonucleases O and Q, the two putative nucleolytic activities which we detected previously in the crude extract from a thermosensitive ribonuclease P mutant (TS241) of Escherichia coli and which were shown to function in the processing of tRNA precursors in vitro, were partially purified from the 100000 ×g supernatant fraction of E. coli Q13. In the course of purification of these enzymes, the total RNAs synthesized in the thermosensitive mutant at the restrictive temperature were used as the substrates and the activities were identified from disappearance or alteration of specific tRNA precursor molecules in polyacrylamide gel electrophoresis. The purified ribonuclease O preparation cleaved specifically the multimeric tRNA precursors at the spacer regions. The purified ribonuclease Q preparation removed, in accordance with the definition of this enzyme, extra nucleotides from the 3′‐terminal ends of monomeric tRNA precursors. Some properties of these two nucleases were investigated. In addition to these nucleases, another exonuclease (tentatively designated ribonuclease Y) and ribonuclease P, a well‐characterized endonuclease, were also purified. The sequential mode of the processing of tRNA precursors, originally observed in the cleavage reactions with the crude extracts in vitro, was supported by studies with the purified enzyme preparations.
Thermodynamic and Kinetic Parameters of Oligonucleotide—Oligopeptide Interactions
Authors: PÖRSCHKE Dietmar
Published: March 2005

It is demonstrated that field‐jump techniques provide a very useful method for the investigation of protein—nucleic acid interactions. Electric field pulses induce the dissociation of nucleotide—peptide complexes by a dissociation field effect. Amplitudes and time constants of this effect can be used to determine both thermodynamic and kinetic parameters. This method, as well as conventional ultraviolet absorbance and circular dichroism titrations, is applied to study the interaction of various combinations of the oligonucleotides (A)4, (A)5, (A)6, (dA)6, (I)6, (U)6 and (C)6 (all lacking terminal phosphates) with the oligopeptides (Lys)2, (Lys)3, (Arg)2, (Arg)3, Lys‐Gly‐Lys, Lys‐Phe‐Lys, Lys‐Tyr‐Lys and Lys‐Trp‐Lys. As demonstrated by chain length dependences the affinity increases with an increasing number of negative charges on the oligonucleotide and of positive charges on the oligopeptide. In addition to these electrostatic effects there are some more specific interactions. This is demonstrated by the fact that the stability constant for the interaction of (I)6 with (Arg)3 is higher by a factor of 4 than the corresponding constant obtained with (Lys)3. This effect is attributed to hydrogen bonding between the guanidino group of arginine and the hypoxanthine base. The data obtained with peptides containing aromatic residues reveal only relatively weak interactions of these residues with the bases of the oligonucleotides. The dynamics of the oligonucleotide—oligopeptide interactions are characterised by very high rates. The complex formation is a diffusion‐controlled reaction with some acceleration due to electrostatic attraction effects. The lifetime of the complexes investigated is in the microsecond time range.
Phosphorylation in vivo of Proteins Associated with Heterogenous Nuclear RNA in HeLa Cell Nuclei
Authors: BLANCHARD Jean‐Marie , BRUNEL Claude , JEANTEUR Philippe
Published: March 2005

As a step towards understanding the role of nuclear ribonucleoprotein particles (hnRNA · proteins) in the nucleocytoplasmic transfer of heterogenous nuclear RNA sequences destined to become the cytoplasmic messenger RNA, the phosphorylation in vivo of the protein moiety of these hnRNA · proteins from HeLa cells has been studied.

After exposure of HeLa cells to [32P]orthophosphate in vivo, purified hnRNA · proteins were found to contain radioactivity covalently bound to proteins in the form of serine and threonine phosphate esters. The association of these phosphoproteins with hnRNA was demonstrated by sedimentation analysis in sucrose gradients, isopycnic banding in cesium chloride density gradients and chromatography on oligo(dT)‐cellulose.

The pattern of radioactive phosphorylated species has been analysed by electrophoresis on sodium dodecylsulphate/polyacrylamide slab gels followed by autoradiography. Although some hottrichloroacetic‐acid‐resistant 32P radioactivity was present throughout the gel, the highest amount of label was found associated with four discrete bands of molecular weights 28000, 30000, 37000 and 52000.

Incorporation of 32P into hnRNA · proteins could be detected after labeling periods as short as 15min, the same species being labeled after 1 and 24‐h labeling times. Although all the above species appear to be present in hnRNA · proteins of different size classes, as resolved by sedimentation through sucrose gradients, their relative proportion is not reproducibly constant.

Exposure of hnRNA · proteins to conditions of increasing ionic strength led to the ready dissociation of the 28000‐Mr species at variance with other labeled species which appeared to be more firmly bound to hnRNA than the bulk of proteins. Upon treatment with pancreatic and T1 ribonucleases, the 28000‐Mr species was also preferentially released while the 37000‐Mr one was the main phosphorylated species remaining associated with core structures containing an RNA component resistant to nucleases which is now under further characterization.
Complete Nucleotide Sequence of the Simian‐Virus 40 Hind‐G Fragment and Localisation of the Carboxyl Terminus of the VP1 Protein
Authors: VAN HEUVERSWYN Hugo , André VAN DE VOORDE  , FIERS Walter
Published: March 2005

The restriction fragment Hind‐G represents 7.0% of the simian virus 40 (SV40) genome. The information present in fragment Hind‐G is expressed as part of the major, late 16‐S messenger RNA. The complete nucleotide sequence of the fragment Hind‐G has now been determined by application of the procedure of Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. (1977) 74, 560–564]. It contains 369 nucleotide base pairs. On the basis of the termination code words in the strand with the same polarity as the late mRNA, two illegitimate reading frames can be defined. Therefore the third, open frame must code for the carboxyl terminal part of the VP1 protein. It terminates within fragment Hind‐G with a TGA signal. This stop codon is followed by a non‐translated region of the mRNA of about 83 nucleotides. The latter contains the sequence A‐A‐U‐A‐A‐A, common to all other eukaryotic mRNA molecules so far studied. The Hind‐G fragment also contains sequences which presumably play a role in the synthesis, processing and/or expression of early mRNA; these aspects are discussed in the following paper.
Streptomycin Uptake via an Inducible Polyamine Transport System in Escherichia coli
Authors: HÖLTJE Joachim‐Volker
Published: March 2005

The rate of uptake of streptomycin by Escherichia coli K12 becomes greatly enhanced after 10–20 min of incubation. Maximal rate of streptomycin and of gentamicin uptake is achieved by preincubation of the cells at 37 °C for 20 min in the presence of 5 μg/ml of streptomycin, kanamycin, gentamicin or neomycin. Spectinomycin and kasugamycin failed to increase streptomycin uptake. Enhanced streptomycin uptake is absolutely energy‐dependent.

The uptake of streptomycin by wild type E. coli is much repressed in the presence of glucose. A cya mutant of E. coli which cannot synthesize adenosine 3′:5′‐monophosphate (cyclic AMP) exhibits poor uptake of streptomycin. The addition of cyclic AMP however, induces the uptake of streptomycin in this mutant.

Transport of spermidine is ten times increased in the presence of 5 μg/ml streptomycin. The uptake of putrescine is enhanced two‐fold under the same conditions. Increase of uptake is prevented by chloramphenicol.

Streptomycin competitively inhibits the transport of spermidine and putrescine. Conversely, spermine, spermidine and putrescine inhibit the uptake of streptomycin.

It is concluded from these results that interaction of streptomycin with the ribosomes results in the induction of a polyamine transport system which can be utilized by streptomycin.
Complex Formation between the Adenovirus Type 5 DNA‐Binding Protein and Single‐Stranded DNA
Authors: van der VLIET Peter C. , KEEGSTRA Wilko , JANSZ Hendrik S.
Published: March 2005

The adenovirus‐type‐5‐coded single‐strand‐specific DNA‐binding protein was purified from infected human KB cells and characterized by sucrose gradient centrifugation and gel filtration. The protein has a sedimentation coefficient of 3.3 S and a diffusion coefficient of 4.4 × 10−7 cm/s, which corresponds to a molecular weight of 68000 for the native protein. This is close to the peptide molecular weight of 72000 and indicates that the protein is a monomer. The frictional coefficient ratio is 1.82 which suggests that the DNA‐binding protein is highly anisometric.

The binding of the protein to various nucleic acids was studied by filtration through nitrocellulose filters and sucrose gradient centrifugation. The binding to single‐stranded DNA is a fast process which does not require bivalent cations or sulfhydryl groups and occurs over a broad pH range. The binding to synthetic polydeoxyribonucleotides is at least 10‐fold less efficient than that to natural single‐stranded DNA.

At low protein‐to‐DNA ratios the binding to single‐stranded DNA is cooperative. A maximal protein to DNA ratio of 33 (w/w) can be obtained which corresponds to 1 protein molecule for about 7 nucleotides. Sedimentation and electron microscopic studies indicate that the DNA‐binding protein keeps single‐stranded DNA in an extended configuration. The saturated nucleoprotein complex is slightly more resistant to nuclease digestion than single‐stranded DNA.
Polypeptide Synthesis in Toluene‐Treated Lymphocytes
Authors: BURRONE Oscar R.
Published: March 2005

Normal human lymphocytes stimulated by phytohemagglutinin P, and other mammalian cells were rendered permeable to macromolecules such as poly(U) and proteins, by treatment with a low concentration of toluene. Under this condition, poly(U) translation was more efficient in the permeabilized cells than in 10000 ×g extracts. Such a process occurs inside the treated cells as demonstrated by the fact that [14C]uridine‐labelled ribosomes remain associated with the toluene‐treated lymphocytes even after incubation at 37 °C.

A nuclease from Staphylococcus aureus was able to penetrate the permeabilized cells and to break the polysome‐bound endogenous messenger RNA. However, the protein‐synthesizing machinery inside the toluene‐treated lymphocytes was unaffected by the nuclease, as demonstrated by the unimpairement of polyphenylalanine synthesis when poly(U) was added after the preincubation with the enzyme. These results suggest that the toluene treatment can be considered as an important tool for the study of the synthesis of macromolecules and its regulation in eukaryotic cells.
Covalent Binding of an NAD Analogue to Liver Alcohol Dehydrogenase Resulting in an Enzyme‐Coenzyme Complex not Requiring Exogenous Coenzyme for Activity
Authors: MÅNSSON Mats‐Olle , LARSSON Per‐Olof , MOSBACH Klaus
Published: March 2005

The NAD analogue, N6‐[N‐(6‐aminohexyl)carbamoylmethyl]‐NAD, was covalently bound to horse liver alcohol dehydrogenase in a carbodiimide‐mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled.

The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3–1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess.

It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40000 cycles/h in a coupled substrate assay.

The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50% whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20000 times as high a concentration of coenzyme.
Dual Action of Double‐Stranded RNA in Inhibiting Protein Synthesis in Extracts of Interferon‐Treated Mouse L Cells
Authors: LEWIS John A. , FALCOFF Ernesto , FALCOFF Rebecca
Published: March 2005

We have investigated the mechanism by which double‐stranded RNA (dsRNA) inhibits protein synthesis in cell‐free extracts prepared from mouse L cells which had been pre‐treated with purified interferon. Preincubation of such extracts for a determined period of time causes inactivation of tRNA species and hence an inhibition of protein synthesis with either Mengo RNA or poly(U,C) as mRNA. This inhibition can be overcome by addition of tRNA. We show that dsRNA inhibits the translation of Mengo RNA irrespective of the period of preincubation and that this inhibition is not overcome by tRNA. Translation of poly(U,C) is unaffected by dsRNA. Thus dsRNA does not act at the level of peptide chain elongation and does not cause inactivation of tRNA species, indicating that dsRNA inhibits protein synthesis in extracts of interferon‐treated cells by a mechanism which is different from that responsible for the inhibition observed in the absence of dsRNA.

The addition of dsRNA to extracts prepared from interferon‐treated cells causes two modifications which are expected to inhibit protein synthesis. (a) An enhancement of the level of nuclease activity, causing Mengo [3H]RNA to be degraded more rapidly in interferon‐treated cell extracts than control extracts, even when association of Mengo RNA with ribosomes is inhibited by the addition of pactamycin. (b) An impairment in the formation of methionyl‐tRNA · 40‐S‐ribosomal‐subunit complexes. The reduced binding of methionyl‐tRNA to 40‐S subunits can be overcome by the addition of a crude initiation factor preparation suggesting that dsRNA causes inactivation of an endogenous initiation factor(s).

In the early stages of translation of Mengo RNA (10–15 min) dsRNA causes a reduction in the incorporation of [35S]methionine into the N‐terminal position of polypeptides suggesting that initiation of protein synthesis is impaired, presumably as a result of the block of methionyl‐tRNA · 40‐S‐subunit complex formation. At a later stage (30–45 min) Mengo RNA is almost completely destroyed under the normal conditions of protein synthesis.
The Effect of Fatty Acids on the Regulation of Pyruvate Dehydrogenase in Perfused Rat Liver
Authors: SCHOLZ Roland , OLSON Merle S. , SCHWAB Andreas J. , SCHWABE Ursula , NOELL Christiane , BRAUN Wolfgang
Published: March 2005

The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14CO2 from infused [1‐14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3‐fold at 0.05 mM pyruvate) but decreased the rate to one‐third of control rates at pyruvate concentrations near 5 mM. These effects were half‐maximal at fatty acid concentrations below 0.1 mM. Infusion of 3‐hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations.

The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.
Use of the T4 Polynucleotide Ligase in The Joining of Flush‐Ended DNA Segments Generated by Restriction Endonucleases
Authors: SGARAMELLA Vittorio , EHRLICH Stanislav D.
Published: March 2005

Double‐stranded DNA segments with completely base‐paired ends were obtained by the action of various restriction endonucleases on phage and plasmid DNAs. These segments were joined covalently by the T4 polynucleotide ligase. The joining was monitored by the electron microscopy count of intramolecularly circularized segments. The highest extent of joining, close to 75%, was observed at 15–25°C with the segments resulting from the action of the Bacillus subtilis (strain R) restriction endonuclease Bsu on the DNA of bacteriophage SPPI or of the plasmid pSC 101. The joining of double‐stranded termini required about 10 times more enzyme than the short single‐standed termini produced by the Escherichia coli restriction endonuclease EcoRI. A shortened purification of the T4 ligase was found to give an enzyme devoid of interfering nucleases.
Incorporation and Asymmetric Orientation of Glycophorin in Reconstituted Protein‐Containing Vesicles
Authors: van ZOELEN Everardus J. J. , VERKLEIJ Arie J. , ZWAAL Robert F. A. , van DEENEN Laurens L. M.
Published: March 2005

Glycophorin can be incorporated in lipid vesicles without the use of detergents [MacDonald, R. J. and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206–9214]. In this paper it has been shown that the protein spans the lipid bilayer in these vesicles. In addition it has been shown that the protein is orientated in an asymmetric way with 75–80% of its sugar residues directed to the outside of the vesicles. Freeze‐fracturing electron microscopy suggests that the protein is present in a slightly aggregated form. Sonication of these recombinants leads to the formation of small glycophorin‐containing vesicles, in which almost all sugar residues are directed to the outside.
Cycloheximide Causes Increased Accumulation of Translatable mRNA for Tyrosine Aminotransferase and Tryptophan Oxygenase in Livers of Cortisol‐Treated Rats
Authors: HOFER Erhard , SEKERIS Constantin E.
Published: March 2005

Messenger RNA activities for two cortisol‐inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated.

Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five‐fold and two‐fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still severalfold higher than in control animals. Application of α‐amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs.

The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.
Mechanistic Aspects of the Transfer of Nascent Periplasmic Proteins across the Cytoplasmic Membrane in Escherichia coli
Authors: PAGÈS Jean Marie , PIOVANT Michel , VARENNE Stanislas , LAZDUNSKI Claude
Published: March 2005

Spheroplasts, prepared according to an efficient procedure which is described, secrete a smaller protein fraction of the total cell content than that secreted by intact cells. However, more proteins are found associated to the cell envelopes derived from these spheroplasts than to those derived from intact cells.

This result was confirmed when we measured, by specific immunoprecipitation, the amount of secreted and unsecreted alkaline phosphatase. Whereas at any time, at 30 °C, in intact cells 99.6% of the alkaline phosphatase produced is secreted and found in the periplasm [see A. Torriani (1968) J. Bacteriol. 96, 1200–1207], only 40% was secreted by our spheroplast preparation. We suggest that partial depletion of outer‐membrane‐bound protease (or proteases), involved in processing of precursors of secreted protein, is responsible for the significant defect of secretion of precursors which then remain membrane‐bound. Such an experimental protocol has been carried out at various temperatures and the secretion of alkaline phosphatase was assayed. This secretion was found to slightly vary above 22 °C whereas it dramatically decreased below 22 °C. Practically no phosphatase was secreted at 13 °C. However, the synthesis of alkaline phosphatase was not differentially altered at low temperatures compared to total proteins. The inactive presumed precursor of this enzyme which was then not secreted was found associated to the cell envelope. Similar results have been observed in intact cells above 22°C where only the energy of protein synthesis (ΔE= 79 kJ), which causes the elongation of nascent chains, is used for protein secretion; below 22 °C, in the temperature range of the disorder‐order transition of the lipid paraffin chains associated to membrane, a ΔE of 125 kJ is required for secretion of alkaline phosphatase. A ΔE of 146 kJ was found for this process to occur in the same temperature range in spheroplasts. When oleate was incorporated into the membrane of strain K 1059, an unsaturated fatty acid auxotroph, the same result as with wild‐type strains was observed, but a decrease in secretion at high temperatures was induced by this modification of fatty acid composition of the membrane. When elaidate instead of oleate was incorporated into K1059 membrane, the fall in alkaline phosphatase secretion occurred at 33 °C instead of 22 °C, which indicates that the physical state of the lipid phase is really involved in the secretion process. A hypothetical model for secretion of nascent periplasmic proteins, based on the data reported, is presented.
Evidence for Synthesis of Alkaline Phosphatase on Membrane‐Bound Polysomes in Escherichia coli
Authors: VARENNE Stanislas , PIOVANT Michel , PAGÈS Jean Marie , LAZDUNSKI Claude
Published: March 2005

Polysomes containing nascent chains of alkaline phosphatase have been isolated from a membrane‐bound polysome preparation. Indirect immunoprecipitation using conformation‐specific antibodies has been employed. This technique provides a good enrichment of these polyribosomes since routinely no more than 10–15% of non‐specific immunoprecipitation was observed. The yield of the procedure is generally 40% but can be increased if higher non‐specific immunoprecipitation is tolerated. Antibodies, previously described, directed against uncoiled or folded monomers of alkaline phosphatase can be used as primary antibody to recognize the nascent chains contained in membrane‐bound polysomes which suggests that these chains are partially folded.
Dependence upon pH of Steady‐State Parameters for the β‐Galactosidase‐Catalysed Hydrolyses of β‐D‐Galactopyranosyl Derivatives of Different Chemical Types
Authors: WITHERS Stephen G. , SINNOTT Michael L. , JULLIEN Magali , YON Jeanine M. , VIRATELLE Odile M.
Published: March 2005

The effect of pH upon the β‐glactosidase‐catalyzed hydrolyses of aryl galactosides is essentially similar for each of the three steps of their hydrolysis. It differs markedly from that on the hydrolysis of galactosyl pyridinium salts; these proceed through a ‘non‐bottleneck’ pathway.

While pH increase abolishes the rate of every step of the reaction for aryl galactosides, it favors the first step of hydrolysis of the galactosyl pyridinium salts, which supports the hypothesis that catalysis of these compounds originates largely in non‐covalent interactions.
An Intramolecularly Quenched Fluorescent Tripeptide as a Fluorogenic Substrate of Angiotensin‐I‐Converting Enzyme and of Bacterial Dipeptidyl Carboxypeptidase
Authors: CARMEL Amoes , YARON Arieh
Published: March 2005

The N‐acyltripeptide 2‐aminobenzoylglycyl‐p‐nitrophenylalanylproline was synthesized and applied as a substrate in the assay of angiotensin‐I‐converting enzyme from calf lung and human serum, and of the bacterial dipeptidyl carboxypeptidase from Escherichia coli. This compound belongs to a new class of substrates for proteolytic enzymes, having the general structure F–X–Q in which fluorescence of group F is quenched by intramolecular interaction with the group Q. Enzymatic cleavage of the peptide chain (X stands for one or more amino acid resiudes) generates the unquenched F‐containing derivative and the resulting fluorescence is used for quantitative measurement of the hydrolysis rate. Cleavage of the Gly‐Phe(NO2) peptide bond in the weakly fluorescent 2‐amino‐benzoylglycyl‐p‐nitrophenylalanylproline molecule results in appearance of the 71 times higher fluorescence (λmax= 415 nm) of 2‐aminobenzoylglycine. Continuous recording of the rising fluorescence allows convenient, sensitive and specific determination of the enzymatic activity, applicable to crude enzyme preparations and human serum. The activity of the mammalian enzyme, measured by this method, is enhanced by Cl− ions and inhibited by low concentrations of EDTA and [Asn1, Val5]anmgiotenmsin II. Kinetic measurements showed Michaelis‐Menten behavior, Km= 0.21 ± 0.1 mM and 0.16 ± 0.1 mM for the falf lung and the bacterial enzyme respectively.
Probable Internal Homology in Thyroglobulin Peptide Chain
Authors: MARRIQ Claudine , ROLLAND Marcel , STEIN Anne , LISSITZKY Serge
Published: March 2005

The time course of digestion of porcine thyroglobulin with subtilopeptidase at an enzyme to substrate ratio of 1:200 was analyzed by sodium dodecylsulfate/polyacrylamide gel electrophoresis after reduction with dithiothreitol. Two fragments of Mr 155000and 135000 were formed which showed a precursor‐product relationship, the 135000‐Mr species accumulating between 1 and 4 h of digestion. After purification by elution from electrophoretic gels, these fragments were treeated by CNBr and analyzed by dodecylsulfat/polyacrylamide gel electrophoresis. Both mixtures of CNBr peptides showed a closely similar electrophoretic band pattern. These results suggest the presence in the thyroglobulin chain (Mr about 140000 associated throught the continuity of the chain to a less structured fragment of Mr about 20000.

A 15000‐Mr peptide was purified by Sephadex G‐200 gel filtration from CNBr‐treated thyroglobulin. Its uniqueness was demonstrated by N‐terminal sequence analysis and by tryptic fingerprinting. Using densitometer recording of stained electrophoretic gels in sodium dodecylsulfate, the molar ratio of this peptide was estimated to be 2 mol/mol of 12‐S thyroglobulin chain.

These results are consistent with a protein structure in which two domains of similar size and structure are linked to form a single chain. Thus, the possibility exists that the structural gene for thyroglobulin might have evolved from duplication and fusion of an half‐sized ancestral gene.
Characterization of a Ca2+‐Dependent Protein Kinase in Skeletal Muscle Membranes of I‐Strain and Wild‐Type Mice
Authors: VARSÀNYI Magdolna , GRÖSXHEL‐STEWART Ute , HEILMEYER Ludwig M.
Published: March 2005

Sarcooplasmic recticulum isolated fromI‐strain andwild type mice contains a Ca2+‐dependent protein kinase activity of about 1.2 U/mg respectively. In addition, these mambranes isolated from I‐strainmicecontain 0.18 U/mg protein phosphatase and a very low amount of phosphatase b activity (0.07 U/mg). In thepresence of Ca2+ions, the membrane‐bound I‐strain mice kinase converts phosphorylase b to the a form; activity proportionally increses with phosphate incorporation. The formed phosphorylase a can be dephosphorylated by phosphorylase phosphatase. Both, the rabbit phosphorylase kinase and the membrane‐bound kinase of I‐strain mice phosphorylate troponin. The vesicles of the sarcoplasmic reticulum isolated from both types of mice show essentially identical ATPase activities of about 1.7 and 1.9 U/mg at 10 nM and of about 2.5 and 2.4 U/mg at 1 μM free Ca2+. 4 mM oxalate stimulates the ATPase activities of both types of mice preparations about 2‐fold. The membrane‐bound Ca2+‐dependent protein kinase from trbbit is half‐maximally inhibited by a concentration of anti‐phosphorylase kinase which is 5–10‐forld higher than that needed for half‐maximalinhibition of soluble phosphorylase kinase. This antibody maximally inhibits the membrane‐bound kinase of both types of mice to about to about 30%. Phosphorylates and non‐phosphoryated purified rabbit phosphorylase kinase show the same affinity for anti‐phosphorylase kinase. Fluoresceinisothiocyanate labelled anti‐phosphorylase kinase stains mainly the sarcolemma and intracellular membranes, probhably the sarcoplasmic reticulum of mice or rabbit diaphragm which is in agreement with the isolation of a Ca2+‐dependent protein kinase together with the latter membranes.