bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell Walls, Lipids, and Lipopolysaccharides of Pseudomonas Species
Authors: Wilkinson Stephen G. , Galbraith Lesley , Lightfoot Gordon A. 
Published: March 2005

Cell walls from two EDTA‐sensitive and three EDTA‐resistant pseudomonads grown in nutrient broth have been analysed. Each wall had a composition broadly characteristic of gram‐negative bacteria, although the walls of Pseudomonas pavonacea and P. syncyanea were relatively rich in glycosaminopeptide and loosely‐bound lipid, respectively. Phosphatidylglycerol was the only phospholipid common to all species, although phosphatidylethanolamine was absent only from P. diminuta. The latter species contained the distinctive range of glycolipids and a phosphoglucolipid identified previously in cells grown on nutrient agar. However, the glycolipids and an ornithine‐containing lipid present in some other species (notably P. rubescens) when grown on nutrient agar were not detected. Except in P. rubescens, the major fatty acids in loosely‐bound lipids were C16, C16:1, and c18:1 acids: in P. rubescens, the major components were C15, iso‐Cl5, and C17:1 acids. Cyclopropane acids occurred in the lipids from P. stutzeri.Components of the lipopolysaccharides were identified and determined. The lipid A fractions contained fatty acids, phosphorus, and (except for P. diminuta) glucosamine. The fatty acids included both hydroxy and non‐hydroxy acids. Apart from 2‐hydroxydodecanoic acid (present in P. syncyanea), all the hydroxy acids were β‐hydroxy acids, and 3‐hydroxydodecanoic acid predominated in all species except P. rubescens. Odd‐numbered hydroxy acids were minor components in P. diminuta and P. pavonacea and major components (C13 acids) in P. rubescens. The non‐hydroxy acids consisted of saturated acids (C12 to C14) and αβ‐unsaturated acids (dehydration products from the β‐hydroxy acids).Unusual features of the polysaccharide fractions from the lipopolysaccharides included the occurrence of non‐phosphorylated heptose residues (P. diminuta), the presence of phosphorylated glucose residues and of aspartic acid (P. pavonacea), the probable absence of 2‐keto‐3‐deoxyoctonic acid and the presence of acid‐labile galactose residues (P. rubescens), the virtual absence of glucose (P. stutzeri), and the presence of alanine (P. syncyanea). Amino sugars (glucosamine, galactosamine, and quinovosamine) occurred in the polysaccharides from P. stutzeri and P. syncyanea only. Following chromatography of the partly degraded polysaccharides on Sephadex, the distribution of components between possible side chain and core regions of the polysaccharides could be determined.The possible significance of the results for the sensitivity of the organisms to EDTA and for their taxonomy is discussed.
A Model for the Initiation of Transcription by DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: Schäfer Rolf , Zillig Wolfram , Zechel Kasper 
Published: March 2005

The stable initiation complexes formed by interaction of DNA‐dependent RNA polymerase from Escherichia coli containing initiation factor σ with native DNAs of bacteriophages T4, T5 and T7 have been differentiated into a “labile‐starter” and a “stable‐starter” class. The former exhibits a half‐life of 1 min with T5‐DNA and of 10 min with T4‐DNA under standard conditions, the latter a half‐life of more than 100 min for the first‐order decay in the presence of the strong template‐competitor heparin. The labile class is competed out by core enzyme, the stable is not. The relative amounts of both classes of complexes depend on ionic strength.The stable‐starter class has been subdivided into a “delayed‐starter” complex formed rapidly and already possessing high stability in the presence of heparin but unable to directly initiate and an “immediate‐starter” complex as a first‐order transition product of the delayed‐starter state. From the immediate‐starter complex initiation can proceed immediately. The time for transition of half of the former into the latter complex is 3.5 min with T5‐DNA. The temperature dependence of the equilibrium concentration of the stable‐starter complex reveals a sigmoid shape indicating a highly cooperative event, probably a conformational change in the transition from delayed to immediate starter.From the length‐average chain length of the transcripts of T5‐DNA in vitro and the length of the transcribed fraction of the DNA an approximate number of 14 promoters per DNA molecule was estimated. For T4‐DNA, calculation yields about 6. In excess of RNA polymerase about 27 copies of transcript per T5‐ “promoter” were produced from the stable complex under conditions excluding consecutive initiation by free enzyme and reinitiation. The corresponding value for T4‐DNA is between 1 and 4.On the basis of these results, a model for the initiation of transcription is postulated, in which an initiation site on the DNA (promoter) is subdivided into an clement for primary recognition, the entry site R, an element for the storage of enzyme for immediate initiation, termed storage stretch S, which furnishes space for the stable alignment of a certain number of polymerase molecules, and an element for actual initiation, termed initiator I.
Étude de l'action de sels de cations monovalents sur les particules ribonucléoprotéiques de cerveau de rat
Authors: Stévenin James , Zawislak Richard , Jacob Monique , de Hildwein Georges 
Published: March 2005

The dissociation of the nuclear particles by salts has been studied in order to throw some light upon their structure.The density of nuclear mono‐ and polyparticles in CsCl was progressively increased as a result of the action of increasing KCl concentrations (from 0.4 to 1.0 M). The results indicate that proteins are progressively released from the particles and are not consistent with the existence of an “informofer” stable at high salt concentration.Polyparticles of high sedimentation coefficient were more resistant to the action of salts than smaller ones. Respectively 69, 35 and 22% of 60‐S, 110‐S and 170‐S particles (density in CsCl: 1.40 g/ml) were dissociated with 1 M KCl into complexes of density (in CsCl) higher than 1.60 g/ml. A further increase in salt concentration or in time of contact, substitution of K+ by Na+ or Li+ ions or the replacement of Mg2+ by EDTA had little effect on the dissociation of 110–170‐S polyparticles. Polyparticles were more easily dissociated by salts when they were first treated with RNAase at very low concentrations, leading to only a few cuts of the polynucleotide chains but not to the hydrolysis of poly‐ into monoparticles. Together the results suggest a compact configuration for the polyparticles.
Conformational Studies on Lipoamide Dehydrogenase from Pig Heart
Authors: van Muiswinkel‐Voetberg H. , Veeger Cornelis 
Published: March 2005

NAD+ forms two pairs of spectrally distinguishable complexes upon binding to oxidized lipoamide dehydrogenase. The shape and magnitude of the spectra is dependent on temperature and pH. The flavin‐absorption band at 450 nm is not shifted uniformly. Intrinsic dissociation constants only could be calculated at 430 nm. In 30 mM phosphate buffer pH 7.2 at 25 °C K1d= 50–60 μM, while at 0 °C K1d= 40 μM for the high‐affinity pair; for the low‐affinity pair these values are 200–250 μM and 110–130 μM respectively. The high‐affinity pair is also present at pH 5.6 at I= 0.1 and I= 0.2, the values at 25 °C are respectively 50 μM and 35 μM.
Computer simulation suggests that the activating effect of NAD+ on the NADH : lipoate reductase activity can be explained by assuming that binding of NAD+ shifts the pK‐value of a protonated group at the active centre from 6.2 to 4.9–5.0. This suggestion is supported by the observation that binding of NAD+ at the high‐affinity site is accompanied by liberation of 0.9 ± 0.07 equiv. H+/mol flavin. From titration data it was calculated that for NAD+ binding K1d= 65 μM.
The results indicate that binding of NAD+ at the high‐affinity site, even at partial saturation, shifts the monomer‐dimer equilibrium towards the dimer. Furthermore the difference in reduction state upon reducing the enzyme with NADH at 25 °C and 0 °C is not due to differences in affinity for NAD+, but to conformational changes of the protein around the flavin moiety. It has to be considered that the two active centres of the dimer are not completely independent.
Binding of Ligands to the Inorganic Pyrophosphatase of Bakers' Yeast
Authors: Rapoport Torn A. , Höhne Wolfgang E. , Heitmann Peter , Rapoport Samuel 
Published: March 2005

Binding studies of ligands to the inorganic pyrophosphatase of bakers' yeast were performed by means of the following techniques: flow dialysis, an electrode sensitive to divalent metal ions, difference spectroscopy and kinetic measurements. The effects of tyrosine modification on binding (and vice versa) were also studied.The results were:

There exist per enzyme molecule four binding sites for the divalent metal ions, presumably two per subunit. Two of the four binding sites are identical with the activator sites postulated from previous kinetic experiments. Binding to these sites induces spectral changes which are similar for all the metal ions. The association constants fall in the order Mn2+ > Co2+ > Mg2+. The role of the other binding sites is still unknown.
Calcium pyrophosphate is bound at two sites per enzyme molecule. It is a good analogue to the actual substrates, i.e. pyrophosphate complexes of Mg, Mn, Co, Zn, since it is bound tightly but is not split by the pyrophosphatase. It protects the enzyme effectively against inactivation by nitration of tyrosines with tetranitromethane.
PPi in the absence of divalent metal ions is weakly bound and is therefore a bad substrate analogue. Pi is also only weakly bound. It is concluded that a stable covalent phosphoryl‐enzyme is not formed under the experimental conditions.
Nitration of an active tyrosine residue in the substrate‐binding site weakens the binding of Co2+ to the activator site by a factor of about 6 while the affinity of calcium pyrophosphate to the substrate site probably completely vanishes.
The Denaturation by Urea and Guanidinium Chloride of Trypsin and N‐Acetylated‐Trypsin Derivatives Bound to Sephadex and Agarose
Authors: Gabel Detlef 
Published: March 2005

Coupling of trypsin to cyanogen‐bromide‐activated Sephadex, but not to agarose, results in a stabilization of the enzyme towards denaturation by urea.
Selectively N‐acetylated trypsin derivatives were prepared and coupled to Sephadex.
When a single amino group, which is required for the immobilization reaction, was available for coupling, the insoluble derivative was denatured as the free one.
The availability of two or three amino groups for coupling diminished the rate of denaturation considerably. When more than approximately four amino groups were available for coupling, the insoluble derivatives were fully active in 8 M urea.
Neither the binding to Sephadex nor that to agarose influenced the denaturation of the enzyme by guanidinium chloride.
Differences between the denaturation mechanisms of urea and guanidinium chloride are discussed.
The Bacterial Degradation of 5‐Amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone
Authors: de Frenne Eberhard , Eberspächer Jürgen , Lingens Franz 
Published: March 2005

From the culture medium of bacteria, which utilize 5‐amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone (pyrazon) as sole source of carbon four metabolites could be isolated. These metabolites were identified as 5‐amino‐4‐chloro‐2‐(2,3‐cis‐dihydroxycyclohexa‐4, 6‐diene‐1‐yl)‐3(2H)‐pyridazinone (compound I), as 2‐(5‐amino‐4‐chloro‐3‐oxo‐2,3‐dihydro‐2‐pyridazino)‐cis,cis‐muconic acid (compound II), as 2‐pyrone‐6‐carboxylic acid (compound III) and as 5‐amino‐4‐chloro‐3(2H)‐pyridazinone (compound IV).
Cell‐free extracts of the pyrazon‐degrading bacteria were shown to metabolize 2‐hydroxy‐muconic acid with a stoichiometric release of CO2.
A pathway for the bacterial degradation of 5‐amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone is proposed.
Biological Role of Xanthine Oxidase and Tetrazolium‐Reductase Inhibitor
Authors: Fried Rainer , Fried Lygia W. , Babin Donald R. 
Published: March 2005

The biochemical properties of tetrazolium reductase inhibitor (“reductase inhibitor”) from beef brain and liver were compared with superoxide dismutase (erythrocuprein) from beef erythrocytes, using xanthine oxidase and xanthine as a model system.In all assays the behavior of erythrocuprein and the reductase inhibitor was identical; it is concluded that the tetrazolium reductase inhibitor is a member of the superoxide dismutase class of enzymes. Its possible identity with other proteins of this group remains to be established.Xanthine dehydrogenase is activated by natural or synthetic detergents. These compounds eliminate the enzymatic action of both erythrocuprein and reductase inhibitor. Low levels of cyanide, which do not inhibit xanthine dehydrogenase block superoxide dismutase.The biological role of xanthine oxidase is discussed. We propose that an important function of xanthine oxidase is to provide an ubiquitous source of hydrogen peroxide and superoxide radicals which serve as oxidants for coupled biological oxidations.
Conditions Affecting the Dissociation Reaction in vitro of Rat‐Liver Ribosomes
Authors: von der Decken Alexandra 
Published: March 2005

Subunits were obtained by incubation of ribosomes with cell sap, puromycin, GTP, phospho‐enolpyruvate, pyruvate kinase, subsequent exposure to 0.15 M KCl and 1 mM MgSO4 followed by sucrose density‐gradient separation.Addition of ATP reduced the yield of the subunits but had no influence on their purity as judged by polyacrylamide‐gel electrophoretic analysis of their RNA. Reassociation of the sub‐units to form 80‐S ribosomes and restoration of polyuridylic‐acid‐dependent synthesis of polyphenylalanine was highest when the subunits were prepared by incubation of ribosomes in the absence of ATP with or without puromycin.In the absence of puromycin, the dissociation rate per minute was 8% and 2% of the total at 35° and 25°C, respectively.Dietary protein restriction excerted no influence on the dissociation of the ribosomes incubated with cell sap of liver from rats fed a normal protein diet, while cell sap from protein‐restricted rats had a decreased capacity to dissociate ribosomes of normal rats. These differences were significant per wet weight of liver but not per mg cell‐sap protein, due to a simultaneous decrease in the protein content of the cell.
Hydrolysis of [3H,4C] Phosphatidylethanolamines by Acid Phospholipases A of Subcellular Fractions of Rat Spleen
Authors: Lloveras Jacques , Douste Blazy Louis 
Published: March 2005

Rat spleen exhibits maximal phospholipase A activity at pH 4.5. This activity is due to a phospholipase A1 and a phospholipase A2. After injection of Triton WR 1339, rat spleens were fractionated by differential centrifugation. The mitochondria + lysosomes fraction was further fractionated on a sucrose gradient. Three lysosomal populations were individualized of which the heavier one was sedimented with mitochondria.With phosphatidylethanolamines as substrate, phospholipase A1 and A2 are present in similar ratio in all the subcellular fractions and in the three lysosomal populations.Rat spleen is about 1.3‐fold richer in phospholipase A1 than in phospholipase A2. The fraction from centrifugation at 1000×g for 10 min contains respectively 40% and 44% of the total phospholipases A1 and A2 activities, the mitochondria + lysosomes fraction only 28% and 26%. Free ribonucleoprotein particles have low phospholipases A1 and A2 activities.
Luminescence Quenching in Azurin
Authors: Finazzi‐Agrò Alessandro , Giovagnoli Carlo , Avigliano Luciana , Rotilio Giuseppe , MondovÍ Bruno 
Published: March 2005

The luminescence of oxidized or reduced azurin, an intensely blue copper protein, has been observed. The addition of Cu2+ or Hg2+ reduced the luminescence of the apoprotein to one third of its value while Ag+ reduced it to two thirds. This quenching is explained in terms of tryptophan distorsion produced by the different coordination of these metal ions. A singlet‐singlet internal conversion is postulated as the mechanism for luminescence quenching in all azurin derivatives. Copper is able to bind both the silver‐ and mercury‐treated protein, as shown by electron paramagnetic resonance experiments, without restoring the native blue color. Moreover addition of copper reduced the luminescence in the silver‐treated protein to that of the native protein; if NaCl is now added, the blue color is restored. This NaCl effect is absent in the mercury‐treated apoprotein.These results suggest that the blue color is related to the binding of copper to at least one of the ligands involved in the mercury or silver complexing, most probably a sulfur atom.
Spin‐Labelled Phosphofructokinase and Its Interactions with ATP and Metal·ATP Complexes as Studied by Magnetic‐Resonance Methods
Authors: Jones Robert , Dwek Raymond A. , Walker Ian O. 
Published: March 2005

Rabbit skeletal muscle phosphofructokinase has been specifically modified at its most reactive thiol group with the nitroxide spin label, 4‐(2‐iodoacetamido)‐2,2,6,6–tetramethyl piperidinooxyl. The specific activity and allosteric kinetics are only slightly altered in the modified enzyme.
The electron spin resonance spectrum of the labelled enzyme at pH 7.5, in the absence of stabilising ligands, indicates that the label has moderate mobility, with a rotational correlation time, τR, of 3.3 ns, which is greater than the mobility of the smallest fully active oligomer of 340000 molecular weight (τR= 90 ns).
The spectrum is sensitive to pH and to the binding of ATP and its magnesium and manganous complexes. The binding of the metal complexes produces an immobilisation of the label (τR= 4.7 ns). In addition to this, MnATP causes a 74% quenching of the signal height as a result of dipolar spin‐spin interaction.
At pH 7.5, MnATP and ATP bind competitively to the labelled enzyme with dissociation constants of 540 μM and 1.3 mM respectively. The binding of MgATP is sigmoidal with [S]0.5= 400 μM and a Hill interaction coefficient of 2.0.
A two‐state model is proposed to account for the electron spin resonance spectral changes, in which the cooperative interactions are due to metal‐ATP binding at catalytic sites, and the dipolar interactions are due to metal‐ATP binding at a separate regulatory site closer to the spin label.
The longitudinal relaxation rate of water protons at 35 MHz is not detectably altered by the presence of the spin label on the enzyme. An analysis of enhancement data obtained in the presence of manganous ions and ATP indicates that the enhancement of the ternary complex, ɛt, is unchanged compared with the native enzyme, but that the dissociation constant for Mn ATP increases from 260 μM in the native enzyme to 480 μM in the labelled enzyme.
The quenching of the electron spin resonance spectrum and the enhancement in the ternary complex of enzyme with MnATP have together been used to estimate the manganese‐nitroxide distance.
The longitudinal and transverse relaxation rates of the H‐2, H‐8, and H‐1′ protons in MgATP bound to the spin‐labelled enzyme are increased in comparison with the native enzyme due to the dipolar spin‐spin interactions. This allows the proton‐nitroxide distances to be estimated.
The distances calculated from the dipolar interactions have been used to obtain a self‐consistent three‐dimensional model of the regulatory site in relation to the spin label.
Enzymatic Conversion of Human Blood‐Group‐O Erythrocytes into A2 and A1 Cells by α‐N‐Acetyl‐d‐galactosaminyl Transferases of Blood‐Group‐A Individuals
Authors: Schenkel‐Brunner Helmut , Tuppy Hans 
Published: March 2005

Human O‐erythrocytes, when incubated with UDP‐N‐acetyl‐d‐galactosamine and α‐N‐acetyl‐d‐galactosaminyl transferase prepared from gastric mucosa of blood‐group A1 individuals, first acquired A2‐specificity and, after continued incubation, became agglutinable by anti‐A1 reagents. Group‐H specificity gradually disappeared. When A2‐erythrocytes were subjected to the same treatment, they required a shorter time of incubation than O‐cells to develop A1‐properties. These findings support the hypothesis of low and high densities of A‐specific sites on the cell surfaces of A2‐ and A1‐erythrocytes, respectively.Acetylgalactosaminyl transferase prepared from the gastric mucosa of blood‐group‐A2 individuals was similar to the corresponding enzyme from A1‐individuals in being capable of transferring acetylgalactosamine from UDP‐acetylgalactosamine to water‐soluble H‐substance and of converting O‐erythrocytes into A2‐cells. With H‐substance as the substrate the blood‐group‐A2 enzyme was considerably less active than the A1‐enzyme based on equal weights of mucosal material extracted. When preparations of A1‐enzyme were diluted so as to show a transferase activity towards blood‐group‐H substance equal to that of the A2‐enzyme preparation, there was still a marked difference between the enzymes with regard to O‐erythrocytes, the speed of their conversion into A2‐cells being much less with the blood‐group‐A2 enzyme as compared with blood‐group‐A1 enzyme. These experiments clearly indicate that blood‐group‐A1 and A2 enzymes have different substrate specificities.
The Influence of Glycosaminoglycans on the Formation of Fibers from Monomeric Tropocollagen in vitro
Authors: ÖBrink Björn 
Published: March 2005

Well‐characterized, monomeric, lathyritic tropocollagen and a variety of well‐characterized glycosaminoglycan preparations have been used in turbidimetric studies on collagen fiber formation in vitro. The experiments have been performed under physiological ionic conditions and at 37°C. It has been shown by others that the fibrillogenetic process consists of two stages, a nucleation phase and a fiber growth phase. By addition of polysaccharides before and after the nucleation phase it was possible to differentiate between the effects of the polysaccharides on the nucleation and on the fiber‐growth, respectively. Chondroitin sulphate, dermatan sulphate, heparan sulphate, heparin and proteoglycans of chondroitin sulphate and dermatan sulphate, which at 4°C form complexes with tropocollagen, in most cases accelerated the fiber formation, when they were added before the nucleation phase. This phenomenon was ascribed to an effect on the nucleation process. When added after the nucleation phase chondroitin sulphate, a heparan sulphate fraction and especially the proteoglycans delayed the fiber formation. This observation was ascribed to an inhibition of fiber growth from the nuclei.Hyaluronic acid, which increases the activity of the tropocollagen molecules by steric exclusion, accelerated both the nucleation and the fiber growth processes. Keratan sulphate, which does not form complexes with collagen, had no or a slightly decelerating effect, either when added before or after the nucleation.It could be demonstrated by light‐scattering technique that certain glycosaminoglycans influenced the properties of the collagen fibrils formed during the early fiber formation process.The observation that certain polysaccharides had different effects on collagen fiber formation, when added before or after the nucleation, shows that the state of aggregation (nucleation) of collagen is of prime importance in determining the effects exerted by the polysaccharides. This finding may explain some of the contradictory data in the literature. Chemical and physical heterogeneity of the glycosaminoglycans also influenced their mode of interaction with collagen during fiber formation.