bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on Energy‐Linked Reactions: Modified Mitochondrial ATPase of Oligomycin‐Resistant Mutants of Saccharomyces cerevisiae
Authors: GRIFFITHS David E. , HOUGHTON Raymond L.
Published: March 2005

Oligomycin resistance is expressed at the mitochondrial and submitochondrial levels in oligomycin‐resistant mutants of Saccharomyces cerevisiae. Studies of the mitochondrial ATPase and Pi‐ATP exchange reaction indicate that a modification of the mitochondrial ATPase (ATP‐synthetase) complex is involved. The specific resistance at the mitochondrial level to oligomycin and related inhibitors such as ossamycin, rutamycin and peliomycin and the lack of resistance to other inhibitors of mitochondrial ATP synthetase (triethyltin sulphate, venturicidin, aurovertin, Dio‐9) correlate with whole cell studies and provide further evidence for a specific modification of an inhibitor site on the mitochondrial inner membrane.

Mitochondrial mutants which map at the OLI and OLII loci can be differentiated biochemically by their sensitivity to oligomycin of mitochondrial ATPase and the pH 9.5/pH 6.5 ATPase activity ratios.

Studies utilising F1‐ATPase from wild type and oligomycin‐resistant mutants indicate that oligomycin resistance is due to a modification of a membrane‐bound component of the mitochondrial ATP‐synthetase complex and not to a modification of F1‐ATPase or the oligomycin‐sensitivity‐conferring protein.

Partially purified oligomycin‐sensitive ATPase preparations (Triton X‐100 extracts) retain the sensitivity (resistance) and specificity of the original membrane‐bound system in mitochondria. Purified oligomycin‐sensitive ATPase preparations also retain the specificity of the original membrane preparation but the sensitivity to inhibitors is partially modified. The results support the conclusion that oligomycin resistance is due to modification of mitochondrially coded and mitochondrially synthesised subunits of the oligomycin‐sensitive ATPase (ATP synthetase) complex. There is no correlation of oligomycin resistance with the ergosterol concentration of yeast cells, yeast mitochondria or mitochondrial ATPase.
Initiation of the Replication of Single‐Stranded DNA by Concerted Action of Phage T7 RNA and DNA Polymerases
Authors: SCHERZINGER Eberhard , LITFIN Frank
Published: March 2005

Phage T7 DNA polymerase in cooperation with T7 RNA polymerase can replicate ФX174 single‐stranded circular DNA. For optimal DNA polymerizing activity, the reaction requires a mixture of all four ribonucleoside triphosphates, or ATP and GTP, or high concentrations (1.5 mM) of ATP alone, in addition to the four deoxyribonucleoside triphosphates. With ATP as the only ribonucleotide present, T7 RNA polymerase is able to carry out ФX174 DNA‐directed synthesis of poly(adenylic acid) de novo. The poly(A) chains formed serve as a primer for T7 DNA polymerase. The DNA synthesized under conditions of coupled poly(A)–DNA synthesis is complementary to the template DNA and contains poly(A) sequences covalently attached to it. With Escherichia coli unwinding protein complexed to the template DNA, the poly(A) or RNA‐primer synthesis is strongly inhibited. This is in marked contrast to the stimulatory effect of the unwinding protein on the action of T7 DNA polymerase.
Carnitine Acetyltransferase: the Question of Multiple Forms
Authors: Edwards Yvonne H. , Chase James F. A. , Edwards Michael R. , Tubbs Philip K.
Published: March 2005

Some properties of carnitine acetyltransferase activity in extracts of various animal tissues and in intact mitochondria have been studied. Enzyme activity was found as soluble and membrane‐associated forms which were separated by isoelectric focusing, ion‐exchange chromatography and centrifugation. It was shown that the two forms of enzyme are freely interconvertible and have similar kinetic properties. Intact mitochondria oxidize acetyl‐CoA (+ carnitine) very much more slowly than acetylcarnitine. The results suggest the existence of only a single type of carnitine acetyltransferase.
Bacteriophage‐Induced Ribonucleotide Reductase Systems
Authors: ERIKSSON Staffan , BERGLUND Ove
Published: March 2005

Infection of Escherichia coli with bacteriophage T5 caused a more than 20‐fold increase in ribonucleotide reductase activity in crude cell extracts. The phage induced the formation of a new ribonucleotide reductase and a new thioredoxin.

The T5‐specific ribonucleotide reductase was defined by its ability to accept the T5‐induced thioredoxin as hydrogen donor. However, the enzyme could also accept E. coli thioredoxin as hydrogen donor and it was inhibited by antisera against subunit B2 of E. coli ribonucleotide reductase. The crude phage enzyme preparation studied preferred CTP to CDP and CMP as substrate and required magnesium and ATP for activity. Its behaviour towards allosteric effectors was different from that of the host enzyme.

T5‐induced thioredoxin chromatographed together with E. coli thioredoxin on DEAE‐cellulose but could be separated from the latter by immunoadsorbent chromatography. T5 thioredoxin was reduced by E. coli thioredoxin reductase but could not serve as hydrogen donor of E. coli ribonucleotide reductase.

Bacteriophage T6 induced a new ribonucleotide reductase and a new thioredoxin upon infection. The T6‐induced proteins cross‐reacted functionally and immunologically with the corresponding T4‐induced proteins.

Infection of E. coli B with phage T7 or induction of the lysogens E. coli K‐39 (λ+) and E. coli W‐3350 (λc1857) caused no increase in reductase activity and chromatography of cell extracts gave no evidence for the presence of new thioredoxins.
A Three‐Dimensional Atomic Model of the Murein Layer of Bacteria
Authors: FORMANEK Helmut , FORMANEK Sibylle , WAWRA Heinz
Published: March 2005

X‐Ray diffraction, infrared spectra and density of murein, the rigid component of almost all bacterial cell walls, were measured using gram‐negative (Spirillum serpens) and gram‐positive (Lactobacillus plantarum) strains.

X‐Ray diffraction of dried foils of murein showed Debye‐Scherrer rings, indicating periodicities of 0.93 to 1.00 and 0.44 nm. The comparison of the infrared spectra of chitin and murein showed that their amide I and amide II bands have the same frequencies. With isopycnic density‐gradient centrifugation of murein a density of ϱ= 1.46 g × cm−3 was observed.

Based on these data, on theoretical considerations and on model building the following structure of murein is proposed: the structure of the glycan chains in murein is similar to that of chitin and cellulose. The peptides consisting of alternating d‐ and l‐amino acids have the 2.27 helical conformation. They are linked to the carbohydrate chains through their N‐terminus and form two hydrogen bonds with the sugar residues. This linkage causes an angle of about 150° between the carbohydrate and peptide chains.
γ‐Cystathionase of Rat Liver
Authors: BROWN Fountaine C. , DEFOOR Marie C.
Published: March 2005

1. With 5′,5‐dithiobis(2‐nitrobenzoic‐acid), 8 of the 12–SH groups in native γ‐cystathionase react reversibly, but 4 form stable bonds and the inhibited product (I) can be isolated. The presence of homoserine, one of the substrates, does not affect the formation of I.

2. Cystathionine, at high concentrations, partially relieves the inhibition; at low concentrations, a different inhibited product, or products, (II) is formed. In form I but not II, the enzyme is protected from reduction by borohydride.

3. Dithiothreitol decomposes both I and II and 65 to 85% of the original enzyme activity can be recovered. Dithiol releases the anion, 2‐nitro‐5‐thiobenzoate, from I but not from 11.
The Influence of Methylene Blue on the Respiratory Metabolism of the Reticulocyte
Authors: RAPOPORT Samuel M. , MÜLLER Marianne
Published: March 2005

Methylene blue selectively increases cytosolic oxidations of the rabbit reticulocytes without uncoupling mitochondrial oxidation. The endogenous substrate of methylene‐blue‐catalyzed oxygen consumption is primarily ribose phosphate, which arises from the breakdown of ribonucleic acids and free nucleotides.

In the presence of methylene blue glucose is oxidized to 95%via the oxidative pentose phosphate pathway. The primary reduction product of the oxygen is H2O2, which is removed both by catalase and by the interplay of glutathione peroxidase and glutathione reductase. Thus a respiratory quotient of 1 results as determined by the balance of 14CO2 formation and O2, consumption.
Enzymatic Deacylation of Methionyl‐tRNAfMef Catalysed by Methionyl, Isoleucyl and Phenylalanyl‐tRNA Synthetases
Authors: SOURGOUTCHOV Andrei , BLANQUET Sylvain , FAYAT Guy , WALLER Jean‐Pierre
Published: March 2005

Native and trypsin‐modified methionyl, isoleucyl and phenylalanyl‐tRNA synthetases from Escherichia coli catalyze the deacylation of methionyl‐tRNAtMet. This activity, which is pH dependent, requires the presence of MgCl2 or spermidine which are known to structure the aminoacyl‐tRNA. Investigation of the effects of cognate amino acids on the rate of enzymatic deacylation of methionyl‐tRNAtMet, reveals strikingly different behaviours of native and trypsin‐modified methionyl‐tRNA synthetases.

Kinetic parameters for the deacylation of methionyl‐tRNAtmet catalyzed by native methionyl and isoleucyl‐tRNA synthetases are compared in the presence of MgCl2 and provide identical Km values of 0.2 μM while their V values, are 0.05 and 0.02 s−1, respectively. This indicates similar enzymatic efficiencies per active site and emphasizes the lack of specificity of the reaction.
Preparation of Chromatin
Authors: de POMERAI David I. , BUTTERWORTH Peter H. W. , CHESTERTON C. James
Published: March 2005

Four of the six chromatin preparation methods examined here are shown to produce chromatins whose template properties differ radically from one another. Comparing the template‐activity profiles obtainable with endogenous (i.e. chromatin‐bound) and exogenous form B rat liver RNA polymerases across a range of salt concentrations, it appears that only one of these chromatins resembles isolated nuclei at all closely. A study of RNA product size on formamide gradients further reveals that only in this single case is there any sign of product‐chains larger than 28‐S rRNA. However, all these chromatins contain considerable exonucleolytic RNase activity, more marked at low than at high ionic strengths. In addition, prolonged (30‐s) shearing is shown to cause drastic changes in the template properties of chromatin, as well as solubilizing it by reduction of fragment size.
Transcription of Yeast DNA in vitro
Authors: PONTA Helmut , PONTA Ulrike , KRAFT Volker , WINTERSBERGER Erhard
Published: March 2005

Yeast DNA as prepared by usual procedures is shown to contain a very effective inhibitor of the initiation of the transcription in vitro. The presence of this inhibitor is responsible for the low template activity of yeast DNA observed by several workers. After removal of the inhibitor by CsCl gradient centrifugation, yeast DNA is readily transcribed by the purified RNA polymerases A and B. Some of the characteristics of the transcription reaction such as the effects of divalent cation (Mn2+ and Mg2+) and salt are described. The purine nucleotide used for initiation of RNA chains was determined. While RNA polymerase A initiates exclusively with ATP RNA polymerase B uses both GTP and ATP for initiation but strongly prefers GTP. Preliminary studies on the transcription inhibitor removed from yeast DNA preparations by CsCl gradient centrifugation suggest that this substance is polyphosphate.
A Spectroscopic Approach to DNA Transcription and Protein Binding
Authors: SAROCCHI Marie‐Thérèse , DARLIX Jean‐Luc
Published: March 2005

Poly(purine) · poly(pyrimidine) polymers exist in two forms, one “normal”, the other protonated and called “hysteretic”, both stable at physiological pH. The normal form is well transcribed by Escherichia coli RNA polymerase whereas the protonated form is not. Binding stoichiometries of RNA polymerase and various proteins to poly(dG) · poly(dC) were followed by ultraviolet difference spectroscopy and compared with those obtained with bacteriophage DNAs. The results obtained suggest that the more specific the binding, the more important the hypochromicity is.
Regulation of the Mammalian Pyruvate Dehydrogenase Multienzyme Complex by Mg2+ and the Adenine Nucleotide Pool
Authors: HUCHO Ferdinand
Published: March 2005

The activity of the mammalian pyruvate dehydrogenase multienzyme complex in vitro depends on the composition of the adenine nucleotide pool (energy charge). An increase of the energy charge from 0.5 to 0.8 can cause a decrease of the activity of the complex by 70%.

At high energy charge, i.e. when the amount of AMP and ADP in the adenine nucleotide pool is low, ATP inhibits the pyruvate‐dehydrogenase phosphatase, the enzyme which reactivates the pyruvate dehydrogenase complex by dephosphorylation. ATP causes this effect by binding Mg2+ essential for phosphatase activity, because it binds Mg2+ much more strongly than ADP or AMP.

From the three enzymes of the multienzyme complex requiring Mg2+ as cofactor, pyruvate dehydrogenase, pyruvate‐dehydrogenase kinase, and pyruvate‐dehydrogenase phosphatase, it is shown that only the latter has a Km for Mg2+ higher than the physiological magnesium concentration. Its Km is 20 mM in phosphate buffer. Therefore the variation of the concentration of unbound Mg2+ caused by a variation of the composition of the adenine nucleotide pool (energy charge) in vivo can be meaningful only for the pyruvate‐dehydrogenase phosphatase activity.

The phosphorylation‐dephosphorylation cycle of the pyruvate dehydrogenase complex results in a net consumption of ATP unless this futil cycle is inhibited. At high ATP concentrations (energy charge > 0.5) this inhibition takes place by binding Mg2+ ions.

Adenosine 3′: 5′‐cyclic monophosphate has no effect on the activity of the pyruvate dehydrogenase complex. Controversial reports on this matter by other authors are discussed.
Phage φ80psu+3‐Directed Tyrosine tRNA Synthesis in Escherichia coli: Effects of T4 Phage Superinfection on Tyrosine Suppressor‐Gene Transcription
Authors: RAAB Claudia , GROSS Hans J.
Published: March 2005

Infection of Escherichia coli cells with the transducing phage φ80psu+3 causes phage‐dependent synthesis of su+3 tyrosine tRNA. This report describes the effect of superinfecting T4 phages on the expression of a single E. coli tRNA gene, namely the φ80 phage integrated su+3 tyrosine tRNA gene, and provides evidence that T4 phage‐induced alteration of the DNA‐dependent RNA polymerase is sufficient to stop E. coli gene transcription.

1. The mutual effects of φ80psu+3 and T4 amN82 phages on their multiplication in E. coli BF266 are as follows: simultaneous infection results in a complete mutual inhibition of phage multiplication; T4 amN82 phage superinfection within 45 min after φ80psu+3 infection has the same result. T4 amN82 phage superinfection later than 45 min after φ80psu+3 phage infection does not affect φ80psu+3 phage multiplication. In all cases of superinfection no viable T4 amN82 phages could be detected, whereas the φ80psu+3 phages multiplied or survived without multiplication.

2. The φ80psu+3‐dependent su+3 tyrosine tRNA synthesis is immediately and absolutely inhibited upon T4 amN82 phage superinfection. The cause of this inhibition is not a selective degradation of the φ80 phage‐incorporated su+3 tyrosine tRNA gene, since it could be demonstrated that φ80 phages isolated from lysates of φ80psu+3/T4 amN82 superinfected E. coli cells still carried the suppressor gene.

3. The inhibition of φ80psu+3 phage‐dependent su+3 tyrosine tRNA synthesis upon T4 amN82 phage superinfection was also observed if the superinfection with T4 amN82 occurred during inhibition of protein biosynthesis.

4. Superinfection of φ80psu+3 phage‐infected E. coli BF266 cells with T4 amN82 ghosts did not inhibit φ80psu+3 phage‐dependent su+3 tyrosine tRNA synthesis.

5. The results described above provide evidence that the inhibition of φ80psu+3‐dependent su+3 tyrosine tRNA synthesis upon T4 amN82 phage superinfection is most likely caused by the T4 phage‐induced alteration of the E. coli DNA‐dependent RNA polymerase.
Electron‐Paramagnetic‐Resonance Studies on Nitrogenase
Authors: ZUMFT Walter G. , MORTENSON Leonard E. , PALMER Graham
Published: March 2005

The oxidation‐reduction properties of azoferredoxin, molybdoferredoxin, and the inactive species of molybdoferredoxin, all iron‐sulfur proteins purified from Clostridium pasteurianum, were studied by potentiometry combined with electron paramagnetic resonance spectroscopy at low temperature. The technique, evaluated with spinach ferredoxin for which a midpoint potential of ‐446 mV was determined (pH 7.9, showed that azoferredoxin has a midpoint potential of ‐294 ± 20 mV (pH 7.5). In the presence of MgATP2−, azoferredoxin undergoes a negative shift of 110 mV in its oxidation‐reduction potential. The resulting potential of approximately ‐400 mV is likely to be effective under physiological conditions. The potential shift is consistent with a proposed conformational change of the protein caused by the binding of two molecules of ATP to the protein (dimer). Molybdoferredoxin and its inactive species, which has an incomplete iron‐sulfur and molybdenum centre, have midpoint potentials of approximately −20 mV and −395 mV, respectively, both at pH 7.5. All nitrogenase proteins are characterized by the transfer of one electron per redox step.

Kinetic studies of the reduction of azoferredoxin and molybdoferredoxin by dithionite gave half times of <10 ms and 3–5 min, respectively. Hence, the latter process must be excluded from being part of the physiological reaction. The complex formation of ATP with azoferredoxin is a relatively slow process, which is markedly accelerated by molybdoferredoxin in the reconstituted nitrogenase system. Changes in the electron paramagnetic resonance signals of reconstituted nitrogenase were followed by a rapid‐freeze technique as a function of mixing sequence and ratio of the two proteins. The observed signal changes were independent of the mixing sequence. The steady‐state level of the molybdoferredoxin signal was also independent of the ratio of the two proteins and was less than 15% of the original resonance. The results indicate that the role of the azoferredoxin · ATP complex may not be confined solely to the function of an electron carrier for the nitrogenase reaction.
Studies of the Activity of Subunits of Aspartate 4‐Decarboxylase Immobilized on Sepharose
Authors: IKEDA Sei‐ichiro , FUKUI Saburo
Published: March 2005

The relationship between the subunit structure and the activity of aspartate 4‐decarboxylase from Pseudomonas dacunhae was studied by immobilizing the dissociated enzyme molecules on CNBr‐activated Sepharose, thus preventing them from reassociation. The Sepharose‐bound dissociated enzyme (half the molecular weight of the native enzyme), which was prepared by immobilizing the enzyme in the presence of 1 M guanidine‐HCl, showed a specific activity about 60% of that for the original undissociated enzyme bound on Sepharose. The subunit structure of the immobilized enzyme molecules was confirmed by means of a dissociation procedure in which the immobilized enzyme was treated with 1 M or 5 M guanidine‐HCl. Reassociation and hybrid formation experiments also supported the idea that the original enzyme activity is higher than the dissociated enzyme activity. 2‐Oxoglutarate activated both the Sepharose‐bound undissociated and dissociated enzymes to the same extent as the native soluble enzyme. The affinity between the substrate and the enzyme was altered somewhat by immobilization, but hardly influenced by the dissociation of the enzyme.
Porcine Thyrotropin
Authors: CLOSSET Jean , HENNEN Georges
Published: March 2005

The properties of porcine thyroid‐stimulating hormone and its subunits, which had not yet been fully characterized, are presented.

A new procedure is described for the isolation of the hormone using only ion‐exchange chromatography and gel filtration. The system, presented in details for porcine thyrotropin was also applied with success for the preparation of bovine and human thyrotropins. The porcine hormone prepared either by the new chromatographic procedure or by countercurrent distribution exhibited identical physicochemical and biological characteristics.

The subunits α and β of porcine thyroid‐stimulating hormone were also obtained by a new procedure. In this method, the native hormone was incubated in acidic urea and the chains were than separated by ion‐exchange chromatography. The subunits were submitted to a complete characterization. The amino‐acid composition of the α subunit of porcine thyrotropin was found identical to that of α chain of porcine luteinizing hormone.

Its amino‐terminal sequence was NH2−Thr‐Met‐Glx indicating that the α‐chain of porcine thyrotropin is shorter by six amino acids than its bovine counterpart.

The porcine β subunit differs markedly in its amino‐acid composition from the bovine β chain particularly with respect to lysine, histidine, tyrosine, threonine and glutamic acid. Phenylalanine is the amino‐terminal residue of the polypeptide chain. The carbohydrate compositions of the two subunits are presented. Porcine thyrotropin differs from its bovine counterpart in that it contains sialic acid, present only in the α subunit.
Metabolism of Trehalose in Euglena gracilis
Authors: BELOCOPITOW Enrique , MARÉCHAL Luis R.
Published: March 2005

Phosphoglucomutase for β‐glucose 1‐phosphate, an enzyme present in cell‐free extracts of Euglena gracilis var. bacillaris, catalyzes the reversible conversion of β‐glucose 1‐phosphate to glucose 6‐phosphate. It was purified 460‐fold by treatment with protamine sulphate, gel filtration in Sephadex G‐100 and chromatography on a DEAE‐cellulose column. The optimum pH of the reaction was 7.0 and the equilibrium constant β‐glucose 1‐phosphate/glucose 6‐phosphate was 0.035. The enzyme has an absolute requirement for β‐glucose 1,6‐bisphosphate as well as a bivalent cation such as Mg2+, Co2+ or Mn2+. Measurements in Sephadex G‐100 gave an apparent molecular weight of about 27000.

This enzyme together with a trehalose phosphorylase found in the same Euglena extracts would constitute a new catabolic pathway for trehalose.

The functions of α‐ and β‐glucose 1,6‐bisphosphate as regulation factors in the energy furnisher system in Euglenais discussed.
Characterization of an Intracellular Protease Isolated from Bacillus thuringiensis Sporulating Cells and Able to Modify Homologous RNA Polymerase
Authors: LECADET Marguerite‐M. , LESCOURRET Monique , KLIER André
Published: March 2005

The level of intracellular proteolytic activities was measured in Bacillus thuringiensis sporulating cells and it is shown that the maximum is reached after 4 h from onset of sporulation (t4). A purification procedure using cells harvested at t5 is reported, which leads to the isolation of an intracellular protease totally different from the extracellular enzymes and from another intracellular fraction detected in crude extracts.

The homogeneity of the purified enzyme was verified using immunological techniques, electrophoretic migration and kinetics of thermodenaturation. Its molecular weight was estimated to be about 23000. This enzyme was characterized as a seryl protease, totally inhibited by phenylmethylsulfonyl fluoride or EDTA and requiring Ca2+ ions for activity. From the esterolytic activity of the enzyme we deduce a chymotrypsin‐like specificity.

The pure protease was able to cleave specifically the β' subunit of the form II of B. thuringiensis RNA polymerase purified from t5 sporulating cells. The vegetative holoenzyme and core enzyme appear much less susceptible to the action of the protease. Moreover the σ and α subunits do not seem to be altered by similar treatments. The existence of a mechanism regulating the level of intracellular proteases during the sporulation phase is postulated.
Construction, Isolation and Implications of Repressor‐galactosidase ·ß‐galactosidase Hybrid Molecules
Authors: KANIA Jürgen , MÜLLER‐HILL Benno
Published: March 2005

Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor‐galactosidase and the enzyme β‐galactosidase, were constructed. Repressor‐galactosidase. in which fully active lac repressor is covalently linked to active β‐galactosidase, is an aggregate with a core structure of four β‐galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric β‐galactosidase, retain all the biological activities of tetra‐meric lac repressor. Substitution of repressor‐galactosidase subunits with β‐galactosidase subunits leads to hybrid molecules with yβ‐galactosidase subunits aggregated with (4‐y) repressor‐galactosi‐dase subunits (where y= 1, 2 or 3). A 2:2 hybrid, i.e. a tetrameric β‐galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 32P‐labelled λplac DNA than pure lac repressor or repressor‐galactosidase.

The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.
Studies on the Transcription Complex of Escherichia coli RNA Polymerase
Authors: ROHRER Hermann , ZILLIG Wolfram
Published: March 2005

To study the chain elongation phase of enzymatic RNA synthesis ternary transcription complexes with T7 DNA or poly[(dA) · (dT)] as template were isolated by gel exclusion chromatography. The DNA in these complexes contains single‐stranded regions which are recognized by a single‐strand‐specific nuclease from Neurospora crassa. The non‐codogenic DNA strand in the poly[(dA) · (dT)] ternary complex is preferentially hydrolysed by the nuclease. The polymerase protects predominantly the codogenic strand in this complex from digestion by DNAse I. In the T7 DNA ternary complex a DNA fragment with a chain length of approximately 26 nucleotides and an RNA fragment of about 22 nucleotides are protected by polymerase from digestion by DNAse and RNAse.
Kinetics of Inhibition of Deoxynucleotide‐Polymerizing Enzyme Activities from Normal and Leukemic Human Cells by 9‐β‐D‐Arabinofuranosyladenine 5′‐Triphosphate and 1‐β‐D‐Arabinofuranosylcytosine 5′‐Triphosphate
Authors: DICIOCCIO Richard A. , SRIVASTAVA B. I. Sahai
Published: March 2005

Both 9‐β‐D‐arabinofuranosyladenine 5′‐triphosphate (aATP) and 1‐β‐D‐arabinofuranosylcytosine 5′‐triphosphate (aCTP) inhibit terminal deoxynucleotidyl transferase and DNA polymerases α and β but not DNA polymerase γ from human cells. Inhibition of terminal deoxynucleotidyl transferase by both compounds is competitive with respect to either dGTP, dCTP, dATP, or dTTP and inhibition of DNA polymerases α and β is competitive with respect to dATP (for aATP) and dCTP (for aCTP). The inhibition constants for aATP for each enzyme are lower than the inhibition constants for aCTP implying that aATP has a stronger affinity for each enzyme than aCTP. In addition, the inhibition constants for aATP for each enzyme are about equal, suggesting that the drug binds to each enzyme with about the same affinity. The same is true for aCTP. Both compounds are non‐competitive inhibitors of terminal deoxynucleotidyl transferase with respect to initiator [(dA)12–18], uncompetitive inhibitors of DNA polymerases α and β with respect to template/primer (activated DNA), and non‐competitive inhibitors of DNA polymerases α and β with respect to a non‐analogous substrate (dTTP). Inhibition of all three enzymes was reversed by simple dilution but not by additional enzyme, template/primer, initiator, or divalent cation. These results suggest that aATP and aCTP inhibit terminal deoxynucleotidyl transferase by competing with any substrate (dGTP, dCTP, dATP, or dTTP) for binding to the enzyme and DNA polymerases α and β by specifically competing with their analogs (dATP or dCTP) for binding to these enzymes.
Interrelation between Transfer RNA and Amino‐Acid‐Activating Sites of Methionyl Transfer RNA Synthetase from Escherichia coli
Authors: JACQUES Yannick , BLANQUET Sylvain
Published: March 2005

Binding of tRNAfMet to the monomeric trypsin‐modified methionyl‐tRNA synthetase turns off the methionine‐dependent isotopic ATP‐PPi exchange. In the case of the dimeric native methionyl‐tRNA synthetase, one anticooperatively bound tRNAfMet inhibits the exchange by only 50%. These behaviours of tRNA do not require the integrity of the 3′‐terminal adenosine. Esterification by methionine of the 3′ end of tRNA reinforces the affinity of tRNAfMet for the enzymes. In the case of the native enzyme, due to this effect, a second binding mode for methionyl‐tRNA may be demonstrated through the isotopic exchange. This additional binding of tRNA corresponds to the expression of the anticooperatively blocked tRNA binding site.

Methionine reverses competitively the reinforcing effect of the esterified methionyl moiety on tRNA binding. It is concluded that after esterification of tRNA, the aminoacyl residue still binds the enzyme, probably within the methionine activating site. The latter behaviour may account for the observation that excess methionine accelerates the aminoacylation turnover rate of tRNAfMet.
Some Characteristics of Phospholipase C from Bacillus cereus
Authors: OTNAESS Anne‐Brit , LITTLE Clive , SLETTEN Knut , WALLIN Reidar , JOHNSEN Sven , FLENGSRUD Ragnar , PRYDZ Hans
Published: March 2005

The amino acid composition of purified Bacillus cereus phospholipase C is reported. The enzyme contains one methionine residue and two fragments are obtained after cyanogen bromide cleavage. The sequence of the amino‐terminal fragment (25 residues) is reported.

Antisera were raised against the enzyme and purified by affinity chromatography. The antisera were monospecific and gave one precipitation line with purified as well as with crude phospholipase C, showing that no antigenic contaminants were present in the purified preparations used as antigen. The antibodies were purified to the extent that about two molecules neutralized one enzyme molecule.

The enzyme is quite resistant to denaturation by urea, sodium dodecyl sulphate or heat (in the presence of 1 mM Zn2+).

Phospholipase C hydrolyses phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Under the conditions used phosphatidylglycerol, cardiolipin, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine and lysophosphatidyl ethanolamine were not substrates. Replacement of Zn2+ by Co2+ or Ni2+ or variation of pH (7.2–8.3), did not change the range of substrates. Phosphatidylcholine was the best substrate among the isolated phospholipids and dicaproylphosphatidylcholine was clearly a better substrate than dipalmitoylphosphatidylcholine.
On the Distribution and Packing of RNA and Protein in Ribosomes
Authors: SERDYUK Igor N. , GRENADER Arkadiy K.
Published: March 2005

From the analysis of the measured radii of gyration of the RNA (Rg= 6.6 ± 0.3 nm) and protein (Rg= 10.2 ⊥ 0.5 nm) components of the 50‐S subparticle of Escherichia coli ribosomes it is concluded that proteins containing a large amount of hydrodynamically bound water are located on the periphery of the tightly packed RNA. We found that the common features of the measured X‐ray scattering curves of the E. coli 70‐S ribosome, its 30‐S and 50‐S subparticles and wheat 80‐S ribosomes in the region of scattering angles corresponding to scattering vectors μ from 1 to 5 nm‐1 reflect features of the RNA compact packing. A hypothesis is proposed that the compact packing of RNA helices in the range of Bragg distances of 4.5–2.0 nm is a general structural feature of all ribosomal particles.
Study of 30‐S Ribosomal Subparticle Protein‐Deficient Ribonucleoprotein Derivatives by X‐Ray Diffusion‐Scattering
Authors: SERDYUK Igor N. , GREMADER Arkadiy K. , KOTELIANSKY Victor E.
Published: March 2005

Treatment of the 30‐S ribosomal particles by 2.15 M LiCl in the presence of 5 mM MgCl2 results in ribonucleoprotein particles which have a sedimentation coefficient of 25.5 S, are capable of being reconstituted to native 30‐S subparticles, and contain 40–50% of the protein bulk of 30‐S subparticles. A comparison of hydrodynamic and X‐ray data for 30‐S and 25‐S ribonucleoprotein particles suggests that the 25‐S ribonucleoprotein particle retains on the whole the structure of the original 30‐S subparticles and therefore the absent proteins (S1, S2, S3, S5, S9, S10, S12, S14, S20, S21 and possibly S11 and S13) do not take part in maintaining the compact structure of the small subparticle of Escherichia coli ribosomes.
Putrescine‐Sensitive (Artifactual) and Insensitive (Biosynthetic) S‐Adenosyl‐L‐Methionine Decarboxylase Activities of Lathyrus sativus Seedlings
Authors: SURESH M. Rangarajan , ADIGA P. Radhakantha
Published: March 2005

The crude extracts of 3‐day‐old etiolated seedlings of Lathyrus sativus contained two S‐adenosyl‐L‐methionine decarboxylase activities. The artifactual putrescine‐dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S‐adenosylmethionine, decarboxylated L‐lysine, L‐arginine, L‐ornithine, L‐methionine and L‐glutamic acid to varying degrees. The decarboxylation was not metal‐ion dependent.

The biosynthetic S‐adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+ ‐dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4‐bromo‐3‐hydroxybenzoylamine dihydrogen phosphate and methylglyoxal‐bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40‐fold by employing a p‐hydroxymercuribenzoate/AH‐Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.
The Effect of Modification of Cytosines in Escherichia coli 16‐S rRNA on Reconstitution and Function of 30‐S Ribosomes
Authors: WREDEE Andrzej , PERZYŃSKI Stanislaw , KLITA Stefan , ZAGÓRSKA Ludwika , SZAFRAŃSKI Przemyslaw
Published: March 2005

O‐Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16‐S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16‐S rRNA chain.

Preparations of methoxyaminated 16‐S rRNA, containing 2–130 modified cytosines/chain, still retained the ability to bind 30‐S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16‐S rRNA.

The particles obtained sedimented slower than native 30‐S subunits, lost their ability to associate with 50‐S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16‐S rRNA did not affect binding of poly(U) by reconstituted particles.
The Location of Secondary Structure in Histone H4
Authors: CRANE‐ROBINSON Colyn , HAYASHI Hiroaki , CARY Peter D. , BRIAND Gilbert , SAUTIÈRE Pierre , KRIEGER David , VIDALI Giorgio , LEWIS Peter N. , TOM‐KUN Jean
Published: March 2005

A total of eight peptides cleaved from calf thymus histone H4 have been studied at several ionic strengths by circular dichroic, infrared and nuclear magnetic resonance spectroscopies to follow the formation of α helix, β structure and self‐aggregates. The results are compared with data obtained previously on three other peptides and on the intact molecule in order to define the location of secondary structure in histone H4. It is concluded that there are two α‐helical sections, the first from residues 55 to 67 and the second of about 12 residues in the region between residues 70 and 90. β‐Structure formation takes place only in the C‐terminal part of the intact H4 molecule. Nuclear magnetic resonance studies of the peptides prove that it is the basic N‐terminal regions of histone H4 that remain free when the molecule self‐aggregates.
Effect of Abrin on Peptide Chain Initiation
Authors: SKORVE Jon , ABRAHAM Abraham K. , OLSNES Sjur , PIHL Alexander
Published: March 2005

Cell‐free systems from wheat germ were preincubated before and after addition of immunoglobulin mRNA to generate systems dependent and independent on peptide chain initiation, respectively. When abrin A chain was added, the system dependent on peptide chain initiation was much more strongly inhibited than the system measuring primarily chain elongation.

Sucrose density gradient analysis showed that abrin A chain did not affect the binding of labelled methionine or labelled mRNA to the 40‐S subunit. However, it caused a strong reduction in the amount of labelled mRNA present in the 80‐S and polysome region. The results indicate that abrin A chain interferes with initiation of protein synthesis by inhibiting the binding of the 40‐S initiation complex to the 60‐S subunit to form the 80‐S initiation complex.

The inhibiting effect of abrin A chain on poly(U)‐directed synthesis of polyphenylalanine was partly overcome by increasing the Mg2+ concentration. A similar effect of Mg2+ was found also in a cell‐free system from Krebs II ascites cells under conditions where primarily chain elongation was measured. Experiments with ribosomes treated with abrin A chain at different concentrations of MgCl2 and then tested for their ability to support polymerization of phenylalanine showed that high magnesium concentrations do not protect the ribosomes against the attack of abrin A chain. The data suggest that high Mg2+ concentrations tend to reverse toxin‐induced conformation changes in the ribosomes.
Transcription Specificity of Yeast RNA Polymerase A
Authors: VAN KEULEN Harry , RETÈL Jan
Published: March 2005

In order to establish more extensively the transcription specificity in vitro of yeast RNA polymerase A, both heterologous DNA's (T7 DNA, linear and circular φ80 DNA, and circular ColE1 plasmid DNA) and total homologous nuclear DNA were used as templates. The transcription products made in vitro were further characterized. The following observations were made.

Yeast RNA polymerase A is only able to transcribe the heterologous templates when single‐stranded breaks or regions are present. These artificial initiation sites, however, are mainly used by the enzyme when relatively high enzyme‐to‐DNA weight ratios are applied.

Using low enzyme‐to‐DNA weight ratios, about 85% of the total cRNA, synthesized in vitro on total homologous nuclear DNA after relatively long incubation times, is derived from the ribosomal DNA fraction, which in yeast accounts for approximately 10% of the total nuclear DNA.

Of the two types of sequences present in yeast ribosomal DNA, i.e. the ribosomal transcription units and the presumably non‐transcribed spacer sequences, the 42‐S precursor cistrons are transcribed completely, whereas virtually no transcription of the spacers takes place.

The order of transcription of the rRNA sequences is first 17‐S followed by 26‐S rRNA. Before reading into the 17‐S rRNA sequences, yeast RNA polymerase A transcribes a stretch of DNA, which has a length of approximately 106 (single‐stranded molecular weight) and specifies a long non‐conserved sequence of the 42‐S rRNA precursor. This is in complete agreement with the well established topographical arrangement of these sequences in the ribosomal transcription unit of yeast.

The maximal length of the transcripts never exceeds an Mr of about 3 × 106, equal to the approximate length of the 42‐S precursor rRNA, even if DNA of a very high integrity is used as a template.

These findings show that yeast RNA polymerase A in vitro has a very strong preference for transcription of its homologous 42‐S ribosomal transcription units. Moreover, our results indicate that initiation and termination of transcription in vitro occur at the correct sites of the 42‐S precursor cistrons.
Transcriptional and Post‐transcriptional Defects in β0‐Thalassaemia
Authors: COMI Paola , GIGLIONI Barbara , BARBARANO Luciana , OTTOLENGHI Sergio , WILLIAMSON Robert , NOVAKOVA Magda , MASERA Giuseppe
Published: March 2005

Complementary DNA enriched in sequences hybridizing to β‐globin mRNA was prepared with viral RNA‐dependent DNA polymerase and used as a probe for the presence of β‐globin mRNA in nuclear and cytoplasmic RNA from two Italian patients with β°‐thalassaemia. In both cases the β‐globin gene was present and cytoplasmic mRNAβ was absent; however, one case appeared to transcribe mRNAβ and to fail to process it, while the other appeared transcriptionally defective. Evidence is also presented that the low levels of hybridization usually found at high RNA/cDNAβ ratios in β°‐thalassaemia are due to δ‐globin mRNA; the melting profile of the hybrid formed has been determined and a low melting temperature relative to mRNAβ· cDNAβ demonstrated.
The Palmityl Binding Sites of Fatty Acid Synthetase from Yeast
Authors: SCHRECKENBACH Thomas , WOBSER Harald , LYNEN Feodor
Published: March 2005

Fatty acid synthetase was covalently labelled with [14C]palmitic acid from [14C]palmityl‐CoA. Tryptic and peptic digestion of the [14C]palmityl enzyme resulted in the formation of radioactive palmityl peptides carrying the long‐chain acyl residue both in oxygen‐ester and thio‐ester linkage. The lipophilic palmityl peptides were purified by column and thin‐layer chromatography using organic solvent systems. peptides arising from the acyl carrier protein, the condensing enzyme and the palmityl transferase were identified and characterized. The amino acid sequence of a 4′‐phosphopantetheine‐containing peptide was established. It comprises 13 residues and shows a high degree of homology with the acyl carrier protein from Escherichia coli. A heptapeptide and an octapeptide from the palmityl transferase active site were partially sequenced. The identical amino acid composition of palmityl transferase and malonyl transferase core peptides is briefly discussed.
Adsorption Equilibria of Thyroid Hormones in the Liver Cell
Authors: WAHL Richard , GEISELER Dietrich , KALLEE Ekkehard
Published: March 2005

The adsorptive distribution of L‐thyroxine and L‐triiodothyronine at 0.04–25 nM concentrations was determined in suspensions of mitochondria in soluble cytoplasmic proteins from rat livers, at protein concentrations of 0.5–4.7 mg total protein/ml suspension. In the concentration ranges measured no saturation of both mitochondria and soluble proteins with thyroid hormones could be observed. The distribution of the hormones between the liquid phase of cytoplasmic proteins and the solid phase of the mitochondria depended mainly on the ratio of the amounts of proteins in both phases.

Certain drugs such as chlorpromazine and phenobarbital in concentrations used therapeutically can interfere with the processes of adsorptive transport in vitro. Chlorpromazine, in the presence of desorptively functioning cytoplasmic proteins or serum proteins, promotes the adsorption of thyroxine and triiodothyronine onto the mitochondria. In contrast, phenobarbital weakens the binding of the hormones to the mitochondria.

The processes of adsorptive binding of thyroid hormones to cell proteins in vitro are suggested as possible links in a transport chain between different compartments in vivo. The new model may also explain the effect of drugs on the distribution of thyroid hormones within the cell.
Comparative Studies of Lactate Dehydrogenases in Lactic Acid Bacteria Amino‐Acid Composition of an Active‐Site Region and Chemical Properties of the L‐Lactate Dehydrogenase of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus acidophilus
Authors: HENSEL Reinhard , MAYR Ulrich , FUJIKI Hirota , KANDLER Otto
Published: March 2005

The molecular weight, the amino acid composition and the N‐terminal and C‐terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non‐allosteric (L. plantarum, L. acidophilus) L‐lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined.

The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the substratebinding site of the L‐lactate dehydrogenase of several animals. However, the ‘essential’ cysteine residue 165 is replaced by threonine, as it is in the L‐lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L‐lactate dehydrogenases, but also the allosteric and the non‐allosteric lactate dehydrogenases from bacterial sources may have evolved from a common gene.
Die hämolytischen Eigenschaften der membranmodifizierenden Peptidantibiotika Alamethicin, Suzukacillin und Trichotoxin
Authors: Irmscher G. , JUNG Günther
Published: March 2005

The Hemolytic Properties of the Membrane Modifying Peptide Antibiotics Alamethicin, Suzukacillin and Trichotoxin

The membrane exciting peptide antibiotics alamethicin F‐50, suzukacillin A and B, and trichotoxin A‐40 can be isolated from different strains of the fungus Trichoderma viride. Cultivation, isolation and purification procedures are described in detail for alamethicin F‐50. At micromolar concentrations the peptide antibiotics cause hemolysis in human erythrocytes, while at nanomolar levels they induce the formation of voltage‐dependent pores of multi‐state properties in black lipid membranes.

A detailed study of the hemolytic action with variations of concentration, temperature and incubation time revealed characteristic differences in the mode of hemolysis between the peptide antibiotics, which can be correlated with structural differences between these natural analogs. The addition of bovine serum albumin reduces the hemolytic activity. The lytic action increases considerably from hypertonic to hypotonic media. For comparison the bee venom constituent melittin and sodium dodecylsulfate have been investigated under the same conditions. The lipophilic peptide antibiotics constitute a new class of membrane disrupting agents with particular properties not known from other lytic compounds.

Assuming that the lytic activity is proportional to the concentration of the peptide antibiotic, the temperature dependence of the concentration/activity curves allows the calculation of activation energies of 62 kJ/mol for alamethicin, 67 kJ/mol for suzukacillin A, and 79 kJ/mol for trichotoxin A‐40. However, melittin revealed the very low activation energy of 49 kJ/mol. These values may be correlated with results from circular dichroism and 13C nuclear‐magnetic‐reonance experiments which indicate conformational changes of the peptides aggregating at the membrane/water interphase.