bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of the NAD+: Protein ADP‐ribosyltransferase Responsible for the T4‐Phage‐Induced Modification of the œ Subunit of DNA‐Dependent RNA Polymerase of Escherichia coli
Authors: SKÓRKO Romuald , ZILLIG Wolfram , ROHRER Hermann , FUJIKI Hirota , MAlLHAMMER Reinhard 
Published: March 2005

The NAD+: protein ADP‐ribosyltransferase responsible for the bacteriophage‐T4‐induced modification of RNA polymerase has been purified 100–200‐fold from Escherichia coli B/r infected with the phage T4.The enzyme has a molecular weight around 26000. It does not require magnesium ions and is protected by sulfhydryl reagents. The temperature optimum is around 20°C. The reaction is inhibited at increased ionic strength and by the reaction products nicotinamide and T4‐modified RNA polymerase. The reaction is irreversible. The only reaction products formed to a significant extent in a crude extract are T4‐modified a subunit and one smaller ADP‐ribosylated peptide. As in vivo, the reaction is complete. Both œ subunits in the RNA polymerase monomer are ADP‐ribosylated in one and the same position.The product of modification in vitro appears to be identical with that of modification in vivo. ADP‐ribose has been obtained by hydrolysis both of T4‐modified œ subunit and of the reaction product of egg‐white lysozyme, NAD+ and the transferase responsible for bacteriophage‐T4‐induced alteration of host proteins [Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biochem. 60, 227–238]. The amino acid carrying the ADP‐ribosyl residue appears to be arginine. T4‐modified œ subunit and the major species of T4‐altered œ subunit are identical.An NAD+: protein ADP‐ribosyltransferase using E. coli proteins and egg‐white lysozyme but no RNA polymerase subunit as acceptor(s) has been purified from uninfected E. coli cells.
Substrate Specificity via Ternary Complex Formation with Glutamate Dehydrogenase
Authors: KOEKOEK Henk , ROBILLARD George T. 
Published: March 2005

Very little discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, T2.Upon titration of the binary enzyme‐ligand complex with coenzyme to form the ternary enzyme‐ligand‐coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.
Metastable Aggregates in the Polymerisation of Tobacco‐Mosaic‐Virus Protein
Authors: VOGEL Dieter , DURHAM Anthony C. H. , DE MARCILLAC Guy D. 
Published: March 2005

Metastable ‘overshoot’ aggregates are formed when tobacco mosaic virus A‐protein (4–8 S) polymerises to disks (20–22 S) and stacks of disks (27–30 S) at pH 6.8–7.6, 20–25 °C. Two cases are discernible. First, at ionic strength 0.1 M, below pH 7.1, a transient, definite 26‐S peak appears: it transforms in a matter of hours into stable 22‐S (disk) aggregates, at a rate that decreases with decreasing pH and increasing temperature; above pH 7.1, there are only 4–8‐S and 22‐S aggregates. Second, after a rapid increase of ionic strength from 0.1 to 0.75 M, below pH 7.6 a broad distribution of very large overshoot aggregates forms (maximum at 60 S, at pH 6.9). With decreasing pH, the maximum size of the overshoot aggregates increases and reequilibration to stacks of two disks (28 S) becomes slower. The 4–8‐S and 26‐S aggregates can both form the very large overshoot aggregates, but disks and stacks of disks alone cannot.Under these conditions, disks undergo only limited stacking (27–30 S) and a very slow conformational change. A similar conformational change parallels the decomposition of the very large overshoot aggregates. At pH 8, overshoot does not occur, probably because a higher pH inhibits the growth of rods.Though the structure of the overshoot‐forming aggregates is still unknown, three‐layer aggregates (8 S and 26 S) are one possibility. We present a scheme including all reversible polymerisations so far observed for TMV protein and discuss their implications for the reconstitution of the virus in vitro.
Processive and Synchronous Mechanism of Polynucleotide Phosphorylase Phosphorolysis
Authors: GUISSANI Annie 
Published: March 2005

Polynucleotide phosphorylase is'an exonuclease which shows, under appropriate conditions, two extreme mechanisms of action: progressive or synchronous. In progressive phosphorolysis the enzyme leaves the substrate only when the degradation is complete, whereas in the synchronous mechanism there are as many dissociations as phosphorolysable nucleotides. In order to determine the number of detachments of the enzyme from its substrate, when either the enzyme or the substrate vary, we have undertaken a theoretical study of the phosphorolysis mechanism. We have deduced two series of relationships: on the one hand the percentage, P, of phosphorolysis compared to the percentage of the liberation of the 3′‐OH terminal nucleotide, pN1, for different degrees of progressivity; on the other hand, the respective percentages of liberation of the successive nucleotides pN1 and pN2, independently of the degree of progressivity of the enzymic mechanism.The two series of curves thus obtained relating P to pN1 released and pNi to pNi‐1 released are valid, independent of the length of the substrate considered: comparison of the experimental and theoretical curves relating P to pN1 released enables the determination of the number of detachments of the enzyme under our experimental conditions; comparison of the curves relating pNi to pNi‐1 released permits the location, in a given sequence, of the nucleotides for which the mechanism of phosphorolysis is synchronous or not.The experimental study applied to a synthetic polymer (A)n‐1‐[14C]U shows that the mechanism becomes less progressive when the enzyme is no longer intact.The interpretation of the phosphorolysis of oligonucleotides within the framework of this theo‐retical study leads to the conclusion that in fact the mechanism is simple only in case of homo‐oligonucleotides; with hetero‐oligonucleotides the length, the sequence, the structure and the possible imprint of the original substrate have to be taken into account. We find that, to a first approximation, a synchronous mechanism is obtained with oligonucleotides containing no more than twelve mononucleotides.This study can obviously be generalized to the attack (or the synthesis) of other substrates by exonucleases that react according to either mechanism.
Affinity Labelling of Alcohol Dehydrogenases
Authors: DAHL Knut H. , McKINLEY‐McKEE John S. 
Published: March 2005

dl‐α‐Bromo‐β(5‐imidazolyl)‐propionic acid is a potential affinity labelling reagent for metallo enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis‐Menten‐type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2′‐dipyridyl, 1,10‐phenanthroline and cyclohexanone, which all combine with the active‐site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys‐46, reversible binding is shown to occur at the active‐site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found.
The potential affinity labelling and crosslinking reagent, symmetrical 1,3‐dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis‐Menten‐type at pH 7.0. Several ligands known to complex with the active‐site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo‐first‐order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.
Chick‐Embryo DNA Polymerase γ
Authors: BERTAZZONI Umberto , SCOVASSI Anna I. , BRUN Gilbert M. 
Published: March 2005

The level of DNA polymerase γ as compared to DNA polymerases α and β has been determined in chick embryo by means of specific tests: the amount of γ‐polymerase in the 12‐day‐old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase γ is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment.The γ‐polymerases have been purified from chick embryo nuclei and mitochondria 500–700 times by means of DEAE‐cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase γ is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified γ enzymes are distinct from the chick embryo DNA polymerases α and β and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial γ‐polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.
Physicochemical Parameters and Subunit Composition of Yeast Phosphofructokinase
Authors: KOPPERSCHLÄGER Gerhard , BÄR Jörg , NISSLER Karl , HOFMANN Eberhard 
Published: March 2005

The properties of proteolytically non‐modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be S020,w= 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found.The partial specific volume of the enzyme as determined by three different methods at 20°C is v2= 0.742 ml/g.From equilibrium sedimentation a molecular weight of 835000 ± 32000 was calculated for the native enzyme.Amino acid analysis of the enzyme was performed and the number of half‐cystine residues determined as 11/100000 g is in good agreement with the number of titratable –SH groups after denaturation of the enzyme.The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15% smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate.Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered.
Fate of Histone Messenger RNA in Mengovirus‐Infected Ehrlich Ascites Tumor Cells
Authors: GALLWITZ Dieter , TRAUB Ulrike , TRAUB Peter 
Published: March 2005

Histone mRNA was isolated from mengovirus‐infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a reticulocyte cell‐free protein‐synthesizing system. The amount of translatable histone mRNA decreases during the first hour postinfection by 30%, rises during the following 1–1.5 h by 10–15%, drops progressively in the further course of infection, and reaches 20% of the control at the end of the infectious cycle (8–9 h postinfection). On the basis of the relative histone mRNA contents, the histone‐synthesizing potentials of mengovirus‐infected Ehrlich ascites tumor cells are substantially higher throughout infection than actually expressed in vivo. This result indicates that the virus‐induced shutoff of histone synthesis is not directly the consequence of inactivation or degradation of histone mRNA.Most of the histone mRNA recovered from mengovirus‐infected Ehrlich ascites tumor cells is bound to ribosomes.Late in infection, certain mRNAs are co‐isolated with histone mRNAs, very likely due to loss or shortening of poly(A) occurring after release of the mRNAs from polyribosomes.
Acyl‐Acceptor Specificities of 1‐Acylglycerolphosphate Acyltransferase and 1‐Acylglycerophosphorylcholine Acyltransferase Resolved from Rat Liver Microsomes
Authors: MIKI Yoshinobu , HOSAKA Kohei , YAMASHITA Satoshi , HANDA Hajime , NUMA Shosaku 
Published: March 2005

The acyl‐acceptor specificities of the 1‐acylglycerolphosphate acyltransferase and 1‐acyl‐glycerophosphorylcholine acyltransferase resolved from rat liver microsomes have been investigated with various synthetic 1‐acyl‐sn‐glycerol 3‐phosphate and 1‐acyl‐sn‐glycero‐3‐phosphorylcholine derivatives differing in their fatty acid constituent (myristic, palmitic, stearic, oleic, linoleic or arachidonic acid) as well as with various 1‐acyl‐sn‐glycerophospholipid analogues carrying different polar head groups (phosphorylcholine, phosphoryldimethylethanolamine, phosphorylmonomethyl‐ethanolamine, phosphorylethanolamine, phosphorylethanol or phosphate).
The activities of the two acyltransferases are virtually not affected by the fatty acid constituent of the respective acyl acceptors, except that the 1‐stearoyl and 1‐arachidonoyl derivatives are somewhat less effective acyl acceptors for both enzymes.
The 1‐acylglycerophosphorylcholine acyltransferase utilizes several acyl acceptors differing in the polar head group with the following order of effectiveness: 1‐acyl‐sn‐glycero‐3‐phosphoryl‐choline > 1‐acyl‐sn‐glycero‐3‐phosphoryldimethylethanolamine > 1‐acyl‐sn‐glycero‐3‐phosphoryl‐monomethylethanolamine ∼ 1‐ acyl‐sn‐glycero‐3‐phosphorylethanolamine. 1 ‐Acyl‐sn‐glycero‐3‐phosphorylethanol and 1‐acyl‐sn‐glycerol 3‐phosphate are ineffective. In contrast, the 1‐acylglycerolphosphate acyltransferase is highly specific for 1‐acyl‐sn‐glycerol 3‐phosphate.
The fatty acid constituent of the acylacceptors exerts essentially no effect on the acyl‐donor specificity of each enzyme except in the case of 1‐arachidonoyl‐sn‐glycero‐3‐phosphorylcholine. Furthermore, the acyl‐donor specificity of the 1‐acylglycerophosphorylcholine acyltransferase is virtually not affected by the polar head group of the acyl acceptor.
The results obtained in this work and in our previous investigations support the view that the substrate specificities of glycerolphosphate acyltransferase, 1‐acylglycerolphosphate acyltransferase and 1‐acylglycerophosphorylcholine acyltransferase make an essential contribution to the nonrandom fatty acid distribution in naturally occurring glycerolipids.
Detection of Guanosine‐Nucleotide · Elongation‐Factor‐G Complexes Produced during the Decay of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes
Authors: GIRBES Tomás , VÁZQUEZ David , MODOLELL Juan 
Published: March 2005

The mechanism of formation and decay of complexes containing guanosine nucleotide, elongation factor G (EF‐G) and the ribosome (with or without fusidic acid) has been examined in a purified Escherichia coli system. With a [14C]GDP · [3H]EF‐G · ribosome · fusidic‐acid complex in the presence of either unlabelled GDP plus EF‐G or the antibiotic thiostrepton, it has been found that [14C]GDP and [3H]EF‐G are simultaneously released, which suggests that they dissociate from the ribosome as a binary complex. In fact, guanosine‐nucleotide · EF‐G complexes, which are too unstable to be isolated, seem to be present in mixtures containing either the [3H]GPD · EF‐G · ribosome · fusidic‐acid complex, the [3H]guanyl‐5′‐yl‐methylene‐diphosphonate · EF‐G · ribosome complex, or the [3H]guanyl‐5′‐yl‐imidodiphosphate · EF‐G · ribosome complex, since ribosomes added to these mixtures, together with a 100‐fold excess of the corresponding unlabelled guanosine nucleotide, specifically bind substantial amounts of radioactive guanosine nucleotide.The binary complexes in these mixtures seem to proceed exclusively from the decay of ternary or quaternary complexes, since the same detection procedure fails to reveal binary complexes in mixtures containing only EF‐G and either [14C]GTP, the labelled GTP analogs or [3H]GDP. Moreover, only very small amounts of [3H]GDP · EF‐G complexes are detected in mixtures where either the EF‐G plus ribosome‐dependent GTP hydrolysis or the interaction of [3H]GDP with EF‐G and ribosomes in the absence of fusidic acid is taking place. The results suggest that EF‐G, after binding to the ribosome in the presence of either GTP or its nonhydrolyzable analogs (but not GDP), undergoes a conformational change and retains guanosine nucleotide more strongly. With GTP, the hydrolysis of this nucleotide would reverse this conformational change, facilitating both the release of GDP · EF‐G from the ribosome and the dissociation of this binary complex. Fusidic acid would interfere with the conformational transitions by preserving the prehydrolytic conformation after GTP hydrolysis and/or by inducing it after formation of the GDP · EF‐G · ribosome complex.
Effects of Cations, Antibiotics and Other Agents on the Turnover of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes
Authors: GIRBES Tomás , CAMPUZANO Sonsoles , VÁZQUEZ David , MODOLELL Juan 
Published: March 2005

The turnover of complexes containing elongation factor (EF) G, the ribosome and either GDP plus fusidic acid or guanyl‐5′‐yl methylene diphosphonate or guanyl‐5′‐yl imidodiphosphate has been measured by chasing the complexed, radioactive forms of these guanosine nucleotides with either an excess of unlabelled guanosine nucleotide or the antibiotic thiostrepton. With the three different complexes, the decay promoted by thiostrepton was substantially faster than that induced by the guanosine nucleotide and, under both decay conditions, no simple first‐order kinetics were observed. Assuming that the dissociation of the complexes proceeds via a guanosine‐nucleotide · EF‐G binary complex and a free ribosome, this result suggests that dissociation was reversible and that after it the inactivation of free ribosomes by thiostrepton was faster than the decay of the newly formed binary complex. Increasing the NH4+ concentration from 10 to 180 mM or decreasing the Mg2+ concentration from 30 to 10 mM during measurement of turnover accelerated the chase of [3H]GDP from a [3H]GDP · EF‐G · ribosome · fusidic‐acid complex. Since these variations did not affect the amount of complex present in solution, they evidently accelerated the reactions leading to formation of the complex. In contrast, increasing the NH4+ concentration of the wash buffer used to isolate the complexes on nitrocellulose membranes sharply decreased their recovery.The antibiotics tetracycline, viomycin and several aminoglycosides that induce misreading (streptomycin, neomycin, kanamycin, tobramycin, gentamicin and paromomycin) inhibited the decay of the GDP · EF‐G · ribosome · fusidic acid complex, but other aminoglycosides like spectinomycin and kasugamycin had no effect. Several peptidyl transferase inhibitors (chloramphenicol, lincomycin, viridogrisein, griseoviridin, spiramycin, gougerotin and althiomycin), the EF‐Tu‐inactivating kirromycin and other antibiotics of less well defined mode of action (erythromycin, bottromycin and micrococcin) were also ineffective. Streptomycin and neomycin were effective at concentrations lower than 10 μM thus indicating that they were probably acting specifically and not as a result of a Mg2+ ‐like action caused by their polycationic nature. The antibiotics did not completely block turnover and their effect could be explained by an increase in the affinity of the ribosome for the binary GDP · EF‐G complex. Since all the effective antibiotics bind to the 30‐S subunit and affect functions related to the ribosomal A‐site, the results emphasize the important role of this subunit for EF‐G interaction and suggest that EF‐G binds to the ribosome in a region near the 30‐S portion of the A‐site.A ternary complex containing Phe‐tRNA, EF‐Tu and GTP did not modify the chase rate of [14C]GDP from a [14C]GDP · EF‐G · ribosome · poly(uridylic acid) · fusidic‐acid complex.
Kinetic Studies on the Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in Intact Cells of Neurospora crassa
Authors: HALLERMAYER Gerhard , ZIMMERMANN Richard , NEUPERT Walter 
Published: March 2005

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.In pulse and pulse‐chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside‐binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria.Mitochondrial matrix proteins labelled in pulse and pulse‐chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor‐product type relationship.The results suggest that newly synthesized mitochondrial proteins exist in an extra‐mitochondrial pool from which they are imported into the mitochondria.
Ornithine Decarboxylase Antizyme Is a Normal Component of Uninduced H‐35 Cells and Rat Liver
Authors: HELLER John S. , KYRIAKIDIS Dimitri , FONG Wang F. , CANELLAKIS E. S. 
Published: March 2005

Ornithine decarboxylase antizyme (a non‐competitive protein inhibitor of ornithine decarboxylase induced by the addition of putrescine, spermidine, spermine to a variety of cell lines and rat liver) has been detected in unstimulated H‐35 cells and in normal rat liver. The ornithine decarboxylase antizyme is bound to subcellular components in a nonreactive form from which it can be released as the free antizyme by treatment with high concentrations of salts or low concentrations of diamines or polyamines. Of the latter, putrescine is the most effective; 1 μM putrescine will release free ornithine decarboxylase antizyme from subcellular particles. The molecular weights and general physical properties of the bound ornithine decarboxylase antizyme are the same as those of the induced ornithine decarboxylase antizyme.In H‐35 cells, 80% of the bound form of the ornithine decarboxylase antizyme is localized in the nucleus whereas only 35% of the bound form of the ornithine decarboxylase antizyme is found in the rat liver nucleus. The remaining activity is not associated with ribosomes or polysomes but is localized in a particulate fraction that sediments through a 5–40% sucrose gradient. This bound form of ornithine decarboxylase antizyme also does not react with ornithine decarboxylase unless it is first liberated by polyamines.The presence of ornithine decarboxylase antizyme bound to particles in unstimulated cells and its liberation from the particles by low levels of polyamines as well as its induction in the intact cell by polyamines suggests that it may play a role in modulating ornithine decarboxylase activity in vivo.