bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
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.