bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Subunit Positions within RNA Polymerase Holoenzyme Determined by Triangulation of Centre‐to‐Centre Distances
Authors: STÖCKEL Peter , MAY Roland , STRELL Irmtraud , CEJKA Zdenka , HOPPE Walter , HEUMANN Hermann , ZILLIG Wolfram , CRESPI Henry L. 
Published: March 2005

The complete ‘centre‐of‐subunit structure’ of the multisubunit enzyme DNA‐dependent RNA polymerase was determined by triangulation of the subunit positions using the intersubunit distances calculated from scattering difference measurements and from the corresponding radii of gyration R. In addition to the centre‐to‐centre distances d between the core subunits α2, β and β′ presented in the preceding paper, the values of d between initiation factor σ and α2 (8.4 ± 1.6 nm), β (4.4 ± 2.2 nm) and β′ (10.7 ± 1.5 nm) were derived from R of σ (4.1 ± 0.3 nm) in situ and of the pairs α2‐σ (6.1 ± 0.4 nm), β‐σ (5.6 ± 0.3 nm) and β‐σ (7.5 ± 0.4 nm) within the holoenzyme (α2ββ′σ).The structural parameters of the subunits within their molecular complex are accessible for neutron small‐angle scattering measurements using labelling of the different subunits (deuteration), total reconstitution of isotopic hybrids, scattering length density matching of ‘hydrogenated’ molecular parts and extended exposure times because of weak scattering effects. The overall shape of σ bound to core enzyme (α2ββ′) proved to be identical (within experimental resolution) with σ in the isolated state measured recently by X‐ray small‐angle scattering. The refined shape of isolated σ was reduced to an ellipsoid which was orientated with respect to the core structure (α2‐β‐β′) in a ‘space‐filling’ way around the position of the a centre obtained by triangulation. The complete subunit arrangement of holoenzyme is shown in a three‐dimensional model.
Receptor‐Specific Large‐Scale Purification of Cholera Toxin on Silica Beads Derivatized with LysoGM1 Ganglioside
Authors: TAYOT Jean‐Louis , HOLMGREN Jan , SVENNERHOLM Lars , LINDBLAD Marianne , TARDY Michel 
Published: March 2005

A receptor‐specific affinity chromatographic method for large‐scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, GM1, is hydrolysed to lysoGM1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE‐dextran has been adsorbed and cross‐linked before coupling. Columns of these Spherosil‐DEAE‐dextran‐lysoGM1 beads, in contrast to particles derivatized with lysoGA1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8).
The toxin‐binding capacity was directly proportional to the amount of lysoGM1 in the column: 2.3 mg/μmol lysoGM1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83–107%).
The affinity‐purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G‐100.
Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000‐l cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.
Blood‐Group A and B Determinants are Located in Different Polyglycosyl Peptides Isolated from Human Erythrocytes of Blood‐Group AB
Authors: VIITALA Juha , KARHI Kimmo K. , GAHMBERG Carl G. , FINNE Jukka , JÄRNEFELT Johan , MYLLYLÄ Gunnar , KRUSIUS Tom 
Published: March 2005

The distribution of blood‐group A and B determinants was studied by isolating blood‐group ABH‐active polyglycosyl peptides from delipidated human blood‐group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to Bs I lectin. The glycopeptides bound were further fractionated using the blood‐group‐A‐specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood‐group‐B‐specific isolectin from B. simplicifolia (BsIB4) whereas the Vc‐unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains.The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N‐acetylgalactosamine was found only in the BsI‐bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3‐O‐linked galactose, which was enriched 7.5‐fold in the BsI‐bound glycopeptides and 3,6‐O‐linked galactose, which was also enriched in the BsI‐bound glycopeptides suggesting that these have a more branched structure than the BsI‐unbound glycopeptides.Glycopeptides derived from bands 3 and 4.5 were prepared from A1B‐blood‐group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI‐lectin from both samples. 70% of the BsI‐bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.
Induction of Metallothionein in HeLa Cells by Dexamethasone and Zinc
Authors: KARIN Michael , HERSCHMAN Harvey R. 
Published: March 2005

Metallothioneins are induced by both Zn2+ and dexamethasone in HeLa cells grown in serum‐free medium. Dexamethasone is able to induce metallothionein synthesis in HeLa cells in virtually zincfree medium ([Zn2+] = 0.1 μM). The presence of dexamethasone does not shift the dose/response curve for metallothionein induction by Zn2+, further indicating that the two inducers work through independent mechanisms.Dexamethasone stimulates Zn2+ uptake 1.7‐fold over 24 h. However, there is no increase in Zn2+ uptake during the first 4 h. In contrast, metallothionein synthesis in response to either Zn2+ or dexamethasone is clearly observable within 4 h of exposure to either inducer. The increased intracellular 65Zn2+ content observed at 24 h is completely accounted for by the increased level of metal bound to metallothionein.In a continuous labeling experiment the rate of synthesis of metallothionein reached a steady state after about 4 h, in response to either inducer. The lag period was identical for both dexamethasone and Zn2+, with similarly shaped induction curves. Induction by dexamethasone, but not by Zn2+, was inhibited by progesterone. Zn2+ and dexamethasone appear to induce metallothionein synthesis in HeLa cells by mechanisms independent of one another.
Relationship between the Rate of Citrulline Synthesis and Bulk Changes in the Intramitochondrial ATP/ADP Ratio in Rat‐Liver Mitochondria
Authors: WANDERS Ron J. A. , VAN WOERKOM George M. , NOOTEBOOM Ron F. , MEIJER Alfred J. , TAGER Joseph M. 
Published: March 2005

The relationship between intramitochondrial and extramitochondrial ATP‐utilizing systems and the intramitochondrial ATP/ADP ratio was studied in isolated rat‐liver mitochondria. Citrulline synthesis was used as an intramitochondrial ATP‐utilizing system, and glucose‐6‐phosphate synthesis as an extramitochondrial ATP‐utilizing system. The intramitochondrial ATP/ADP ratio was manipulated in three ways: with succinate and different concentrations of malonate and/or hexokinase; with 2‐oxoglutarate (plus oligomycin) and different concentrations of hexokinase; and with added ATP in uncoupled mitochondria (oligomycin present).
Under all conditions used, citrulline synthesis was strictly correlated with the bulk intramitochondrial ATP/ADP ratio.
The curve relating citrulline synthesis and intramitochondrial ATP/ADP was shifted towards lower ATP/ADP ratios when the activity of carbamoyl‐phosphate synthetase was enhanced by increasing the mitochondrial content of N‐acetylglutamate.
It is concluded that under the experimental conditions used the intramitochondrial adenine nucleotides behave as a homogeneous pool.
Amino Acid Utilisation in Isolated Hepatocytes from Rainbow Trout
Authors: FRENCH Christopher J. , MOMMSEN Thomas P. , HOCHACHKA Peter W. 
Published: March 2005

The utilisation (conversion to CO2 and/or glucose) of a series of amino acids by isolated trout hepatocytes was investigated and compared to the utilisation of lactate and palmitate. In fed fish, several amino acids (alanine, serine, asparagine and glycine) and lactate produced CO2 at considerably higher rates than palmitate. During starvation plus exercise, the rate of CO2 production from palmitate increased while that from lactate and most of the amino acids decreased.Gluconeogenesis from amino acids in fed fish was lower than from lactate. Serine and asparagine were the most effective substrates: alanine gave lower rates of incorporation. During prolonged starvation plus exercise, the rates of gluconeogenesis from amino acids increased twofold and, simultaneously, there was a corresponding increase in phosphoenolpyruvate carboxykinase activity in liver.It is concluded that several amino acids (dietary or released from muscle protein) are potentially major oxidative substrates in trout. In addition, amino acids appear to have the capability to maintain supplies of glucose during a period of prolonged starvation and exercise. No evidence could be found to support the contention that alanine is the most important glucogenic amino acid.
Identification of Different Forms of the Murein‐Bound Lipoprotein Found in Isolated Outer Membranes of Escherichia coli
Authors: WENSINK Jan , WITHOLT Bernard 
Published: March 2005

The identification of the free and murein‐bound forms of theEscherichia coli lipoprotein on dodecylsulphate‐polyacrylamide gels was systematically investigated by analyzing the low‐molecular‐weight proteins (Mr < 20000) of both cytoplasmic and outer membranes.The free form of the lipoprotein was identified on 15% polyacrylamide gels as the fastest migrating component (Mr= 7200–7500) of isolated outer membranes; it could be separated from a small cytoplasmic membrane protein (Mr= 6500) which was probably identical to the dicyclohexylcarbodiimide binding proteolipid of the membrane‐bound ATPase.Lysozyme treatment of both outer membranes and murein sacculi failed to convert the murein‐bound lipoprotein into a fragment of uniform size; instead the bound form appeared as a series of bands consisting of lipoprotein bound to one, two,… eight murein subunits. The composition of this ladder depended on the method used to isolate outer membranes.Beside these lipoprotein bands the outer membrane contained two other proteins, III and V; the relation of these proteins to previously described proteins is discussed.
Effect of NADH on the pKa of Zinc‐Bound Water in Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , LINDSTRÖM Anders , OLDÉN Bertil , PETTERSSON Gösta 
Published: March 2005

Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10–12 by pH‐jump stop‐flow techniques. The binding of NADH or NAD+ requires the protonated form of an ionizing group (distinct from zinc‐bound water) with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pKa of 11.2. The binding of trans‐N,N‐dimethylaminocinnamaldehyde to the enzyme · NADH complex is prevented by ionization of the latter group.It is concluded from these results that the pka‐11.2‐dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The pKa, value of 11.2 thus assigned to zinc‐bound water in the enzyme · NADH complex appears to be typical for an aquo ligand in the inner‐sphere ligand field provided by the zinc‐binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal‐bound water in zinc‐containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active‐site zinc ion.
5‐Aminolevulinic Acid Dehydratase
Authors: SEEHRA Jasbir S. , JORDAN Peter M. 
Published: March 2005

5‐Aminolevulinic acid dehydratase from bovine liver has been shown to be inactivated by 5‐halolevulinic acids and 3‐halolevulinic acids.
The substrate, 5‐aminolevulinic acid, protects the enzyme from modification by 5‐halolevulinic acids.
Using tritiated chlorolevulinic acids, it was shown that four of the subunits in the octameric enzyme are preferentialiy modified.
The susceptible enzyme group modified is an–SH group of a reactive cysteine at or near the active site.
Oxidized enzyme is not affected by either 5‐chlorolevulinic acid or 3‐chlorolevulinic acid.
Evidence is presented which suggests that 5‐chlorolevulinic acid is acting as an active‐site‐directed reagent.
On the Mechanism of Interaction of N‐Acetylphenylalanyl‐tRNAPhe with Ribosomes of Escherichia coli
Authors: IVANOV Yuri V. , GRAJEVSKAJA Rosa A. , SAMINSKY Eugen M. 
Published: March 2005

The tetranucleotide TpΨpCpGp acts as a specific inhibitor of the rate of AcPhe‐tRNAPhe binding in the ribosomal P site. This effect is observed both in the presence and in the absence of poly(U).In the absence of poly(U) antibiotics tetracycline and puromycin also decrease the rate of AcPhe‐tRNAPhe binding. Some inhibition by tetracycline is observed with poly(U).All these inhibitors are known to be ligands of the ribosomal A site, and their influence on the P site binding can be most naturally explained by the suggestion that AcPhe‐tRNAPhe enters the ribosome via the A site, forms there an intermediate complex, and spontaneous translocation into the P site follows. In the presence of poly(U) arguments in favour of this hypothesis are much weaker, but the same sequence of events is possible.
Structure de la région heptose du lipopolysaccharide de Escherichia coli K12 CR34
Authors: BLACHE Denis , BRUNETEAU Maud , MICHEL et Georges 
Published: March 2005

Structure of the Heptose Region of the Lipopolysaccharide from Escherichia coli K12 CR34The heptose region of the lipopolysaccharide of Escherichia coli K12 CR34 was studied. The Glucose linked to the heptose II was found to be substituted by a d‐galactose and the linear chain of the core polysaccharide has two (1→3) linked heptoses. The heptose II is substituted by a lateral (1→7) linked heptose III and the heptose I is linked in (1→5) to 2‐deoxy‐d‐manno‐octulosonic acid. The three sugars of the linear chain, heptose I, heptose II and glucose are substituted by phosphate, pyrophosphate or pyrophosphorylethanolamine group linked to C‐4 hydroxyl groups. However, in some polysaccharidic chains one or two substituting groups may be absent. This result may explain the heterogeneity in the length of the core polysaccharidic chains.
Correlated Effluxes of Adenine Nucleotides, Mg2+ and Ca2+ Induced in Rat‐Liver Mitochondria by External Ca2+ and Phosphate
Authors: ZOCCARATO Franco , RUGOLO Michela , SILIPRANDI Dagmar , SILIPRANDI Noris 
Published: March 2005

The presence of inorganic phosphate and Ca2+ in the external medium induces a closely parallel efflux of both endogenous adenine nucleotides and Mg2+ from rat liver mitochondria. These effluxes are (a) pH‐dependent and inhibited by uncouplers, respiration inhibitors and external Mg2+; (b) completely prevented by bongkrekate, but stimulated by atractylate.ATP, ADP or AMP each inhibit the release of Mg2+ promoted by Ca2+ and phosphate: however, in the presence of oligomycin and P1,P5‐di(adenosine‐5′)‐pentaphosphate (an inhibitor of adenylate kinase) only ADP is effective. Also the release of accumulated Ca2+ observed when ∼ 50% Mg2+ is discharged is retarded by bongkrekate and added Mg2+ whereas it is accelerated by atractylate.All adenine nucleotides have a significant effect in retarding the efflux of accumulated Ca2+ but, in the presence of oligomycin and P1,P5‐di(adenosine‐5′)‐pentaphosphate only ADP is active.From these results we conclude that effluxes of Mg2+, Ca2+ and adenine nucleotide from rat liver mitochondria induced by external phosphate are interconnected and regulated by external ADP and Mg2+ levels.
The Effects of pH and Redox Potential on the Hydrogen Production Activity of the Hydrogenase from Megasphaera elsdenii
Authors: VAN DIJK Cees , VEEGER Cees 
Published: March 2005

The effects of temperature on the ionization constant (pK′) and apparent midpoint potential (EB) of the unprotonated species of Megasphaera elsdenii flavodoxin hydroquinone shows that, above 15° C, ΔpK′· K−1=− 9.7 × 10−3 and ΔEB= K−1=− 0.6 mV. The effects of pH and redox potential on the hydrogen production activity with fixed concentrations of methyl viologen semiquinone (0.3 mM; artificial donor) and M. elsdenii flavodoxin hydroquinone (50 μM; natural donor) show that with decreasing pH the activity increases.Irrespective of the pH and electron donor, at increasing redox potential, a redox‐potential‐independent production activity is followed by a redox‐potential‐dependent production activity. This redox‐potential‐dependent behaviour of the hydrogen production activity represents an n= 2‐type of redox titration curve with an ‘apparent midpoint potential’ which corresponds with the potential of the hydrogen electrode at that pH. The effect of pH on the manometrically determined hydrogen production activity (direct) is in good agreement with that determined spectrophotometrically (indirect; see preceding paper), with both electron donors tested.In contrast to predictions from the models for hydrogenase activity [van Dijk et al. (1980) Eur. J. Biochem. 102, 317–330], a double‐reciprocal plot of the kinetic data for M. elsdenii flavodoxin hydroquinone at pH 5.5 is non‐linear. A slightly adapted kinetic model based on a similar mathematical formulation of its rate equation, to explain the effects of redox potential, proton and electron (donor) concentration on the hydrogenase activity is proposed. This model also explains, on it theoretical basis, the effects of pH and redox potential on the hydrogen production activity.The effect of pH on the hydrogen oxidation activity with methyl viologen and benzyl viologen as electron acceptors shows for both dyes an optimum at pH 9.7. The ratio of the activities with both viologens is constant over the pH range tested.
Binding of Ribosomes to Linear and Circular Forms of the 5′‐Terminal Leader Fragment of Tobacco‐Mosaic‐Virus RNA
Authors: KONARSKA Maria , FILIPOWICZ Witold , DUMDEY Horst , GROSS Hans J. 
Published: March 2005

The sequence of the 5′‐terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61–66] has the 7‐methylguanosine(5′)triphospho(5′)guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues.The RNase‐T1‐resistant 73‐nucleotide‐long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5′ or 3′ end, efficiently formed disome initiation complexes when incubated with wheat‐germ protein‐synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70‐S ribosomes.The results offer further support to the notion that in eukaryotic initiation the free 5′ end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5′ terminus.
Mechanisms for Albumin‐Mediated Membrane Damage
Authors: DRAINAS Denis , HARVEY Elizabeth , LAWRENCE Anthony J. , THOMAS Adedapo 
Published: March 2005

Bee venom phospholipase A2 causes a small increase in the sublytic leakiness of rabbit erythrocytes; which is strongly inhibited by lysolecithin and potentiated by albumin.
Albumin extracts long‐chain fatty acids from the erythrocyte membrane by a process which is faster than diffusion‐determined efflux of fatty acid and during this process it greatly increases the sublytic leakiness of the membrane. In contrast, albumin rapidly terminates sublytic responses to lysolecithin.
These actions of albumin are abolished only when albumin is preloaded with four molecules of oleic acid per protein molecule.
A minor proportion of the response of phospholipase‐A2‐treated cells to albumin is due to accumulated fatty acid products; the combination of active enzyme and accumulated fatty acids is much more effective than either alone. Membrane partially depleted of phospholipids by enzyme attack is not highly leaky.
Albumin may, in part, act by removing lysolecitihin, the inhibitory reaction product. A model is proposed in which susceptibility to phospholipase A2 attack is not uniform on the cell surface and is used to explain the inhibitory action of lysolecithin and the high sensitivity of the membrane in the presence of albumin to damage by newly released fatty acids.
Large‐Scale Purification and Some Properties of Malate Synthase from Baker's Yeast
Authors: DURCHSCHLAG Helmut , BIEDERMANN Gunthard , EGGERER Hermann 
Published: March 2005

Malate synthase from baker's yeast (5 kg) was purified 400–500‐fold to homogeneity. About 50–200 mg homogeneous enzyme were obtained within a week in a yield of 30% with reference to the total activity in cell‐free crude extracts. The enzyme, pl = 7.5, was pure as judged from ultracentrifugal and gel electrophoretic studies.
Sedimentation and diffusion coefficients were determined: . The molecular weight of the synthase was found to be 175000 ± 10000 and 180000 ± 10000 by sedimentation/diffusion and by high‐speed sedimentation equilibrium respectively. It was concluded from these and other results that malate synthase has a molecular mass of 180000 ± 10000.
The synthase on sodium dodecylsulfate gel electrophoresis was dissociated to yield a single specimen of Mr about 66000. The result indicates a composition of the native enzyme from three subunits of identical or nearly identical mass.
The binding of acetyl‐coenzyme A to the synthase is independent of Mg2+ but that of glyoxylate is strictly dependent on the presence of Mg2+. Kinetic studies indicate that the malate synthase reaction follows a sequential random mechanism.
The intermolecular isotopic effect, kH : k2H= 1.4, was determined with acetyl‐coenzyme A and [2H3]acetylcoenzyme A under several different experimental conditions and was shown to reflect different maximal velocities of the two substrates. An enzymic procedure for the preparation of doubly labelled [3H,14C]acetyl‐coenzyme A is also presented.