bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism‐Controlled Stereospecificity
Authors: POLGÁR László , FEJES Judit
Published: March 2005

The activation parameters of acylation of subtilisin with alkyl and p‐nitrophenyl esters of N‐acylamino acid enantiomers were determined. It was found that (1) the activation entropy is much higher with the nitrophenyl esters than with the corresponding methyl esters, (2) the difference in rate constants between enantiomers is 104–105 with methyl esters whereas it is only of the order of 10 with nitrophenyl esters.

The results indicate that the catalytic mechanism is simpler for nitrophenyl esters than for alkyl esters. The simple mechanism requires only general base catalysis, and thus permits more freedom of motion in the transition state, whereas the complex mechanism involves both general base and general acid catalysis. Furthermore, the strikingly low enantiomeric specificity with nitrophenyl esters indicates that not only binding but also the catalytic mechanism is an important factor in determining the stereospecificity of an enzyme.

The activation parameters for enantiomeric nitrophenyl ester reactions suggest that structurally related substrates can be transformed by the enzyme in different conformations which may be energetically similar or not. The energetically different conformations may account for the activation enthalpy‐entropy compensation.
Transport of d‐Lactate in Perfused Rat Liver
Authors: SCHWAB Andreas J. , BRACHT Adelar , SCHOLZ Roland
Published: March 2005

The transport of d‐lactate across the plasma membrane was investigated in hemoglobin‐free perfused rat livers, applying the multiple‐indicator dilution technique (pulse labelling of d‐lactate and indicator substances).

The following results were obtained:

The steady state exchange rate at 1 mM d‐lactate was 2.5 μmol × min−1× g wet wt−1. It was proportional to the extracellular concentration in the range between 0.1 and 70 mM.

The transport of d‐lactate was inhibited by l‐lactate and pyruvate; 50% inhibition was observed at 40 mM l‐lactate or 5 mM pyruvate.

The transport was also inhibited by α‐cyanocinnamate and 4,4′‐diisocyanostilbene‐2,2′‐disulfonic acid. The inhibition by cyanocinnamate was complete (with 25 mM) and fully reversible, whereas the inhibition by diisothiocyanostilbenedisulfonic acid was incomplete and irreversible; it was dependent upon the amount of diisothiocyanostilbenedisulfonic acid bound by the liver. Maximal inhibition (80%) was observed with 2 μmol diisothiocyanostilbenedisulfonic acid bound per g wet weight.

The intracellular concentration (ci) of d‐lactate was proportional to the extracellular concentration (ce); the ratio ci/ce was 0.5 throughout the concentration range studied. It decreased in the presence of l‐lactate or pyruvate.

It is concluded that the transport of d‐lactate is carrier‐mediated, and, at least partially, electro‐neutral.
Dissociation of Thyroglobulin Iodination and Hormone Synthesis Catalyzed by Peroxidases
Authors: VIRION Alain , POMMIER Jacques , NUNEZ Jacques
Published: March 2005

The properties of thyroid peroxidase in the catalysis of thyroglobulin iodination and thyroid hormone synthesis (coupling reaction) were compared to those of horseradish peroxidase. The following results were obtained.

Whereas the thyroid enzyme catalyzes both reactions at the same pH (∼ 7.4) horseradish peroxidase has a pH optimum for iodination of 5.2 and a complex pH dependency for coupling: two peaks of activity were found at pH ∼ 7.5 and ∼ 8.5 with no activity left below pH 5 and above pH 10. These properties of horseradish peroxidase allow a clear dissociation of the iodination and the coupling reactions.

Different experiments were performed in which iodination but not coupling could be obtained with either enzyme; a second incubation of these samples at the same or a different pH and with the same or the other enzyme resulted in different levels of coupling. This procedure revealed the following observations. (a) The low efficiency of horseradish peroxidase in performing the coupling reaction at a low pH is not due to the fact that fewer iodotyrosine residues, potentially able to couple, are formed at this pH. Actually 4 hormonogenic iodotyrosine residues were formed at pH 5.2 with horseradish peroxidase but only two of them were able to couple at this pH. Raising the pH to a more alkaline range resulted in the coupling of these preformed hormonogenic residues, (b) Higher levels of coupling, however, were obtained with horseradish peroxidase only at higher levels of iodination than with thyroid peroxidase suggesting a differential sequence in the iodination by the two enzymes of the different tyrosine residues of thyroglobulin.

The coupling reaction was catalyzed by horseradish peroxidase in the absence of iodide whereas the iodide was required for the thyroid enzyme.
ΔpH Induced Calcium Fluxes in Rat Liver Mitochondria
Authors: BERNARDI Paolo , AZZONE Giovanni Felice , VERONESE Paolo
Published: March 2005

The paper reports an investigation on ΔpH‐driven Ca2+ fluxes in rotenone‐treated rat liver mitochondria. The H+ concentration gradient (acidic inside) required to drive Ca2+ influx is obtained from the K+ concentration gradient through a H+/K+ exchange. The rate of H+/K+ exchange is very low in native mitochondria and is markedly enhanced by the electroneutral ionophore nigericin. The rate of Ca2+ influx depends on the rate of generation of ΔpH; it is negligible in native mitochondria and fast after nigericin.

The process of ΔpH‐driven Ca2+ influx shows three basic features. First, the rate of Ca2+ influx is markedly increased by the addition of uncouplers. Second, the rate of Ca2+ influx is inhibited by ruthenium red with the same sensitivity as the processes driven by the H+ pump or K+ diffusion. The electroneutral H+/Ca2+ ionophore A23187 restores Ca2+ influx in mitochondria inhibited by ruthenium red. Third, both rate and extent of ΔpH‐driven Ca2+ influx are inhibited by the addition of weak bases but not of weak acids.

The process of ΔpH‐driven Ca2+ influx is accompanied by H+ efflux. A correlation also exists between generation of ΔpH (acidic inside) due to H+/K+ exchange and utilization of ΔpH due to H+/Ca2+ exchange. The process of ΔpH‐driven, A23187‐catalyzed, H+/Ca2+ exchange is not preceeded by rapid H+/K+ exchange in the absence of nigericin.

After termination of the phase of Ca2+ influx a phase of Ca2+ efflux ensues. Ca2+ efflux is not coupled to H+ or K+ reentry and is completely inhibited by ruthenium red. Ca2+ efflux is not observed when Ca2+ transport is catalyzed by A23187 in mitochondria inhibited by ruthenium red. Furthermore A23187 abolishes Ca2+ efflux also when Ca2+ influx occurs in the absence of ruthenium red.

The data suggest that the ΔpH‐driven Ca2+ influx and the subsequent Ca2+ efflux take place through the native carrier as electrical uniport.
Crystallization of Bacteriophage MS2
Authors: MIN JOU Willy , RAEYMAEKERS Alex , FIERS Walter
Published: March 2005

Crystals of bacteriophage MS2 have been obtained by slowly cooling a 1% virus solution from 23°C to 0°C in the presence of poly(ethylene glycol) 6000. The crystals were colorless, needle‐like, anisotropic and very fragile. Electron microscopic observation of the crystals revealed a two‐dimensional lattice of particles with RNA phage morphology and dimensions. Preliminary X‐ray examination of the crystals confirmed their viral nature.
Specific Labeling of a Phosphate‐Transporting Protein from Rat‐Liver Mitochondria by [203Hg]Mersalyl
Authors: HOFMANN Hans‐Dieter , KADENBACH Bernhard
Published: March 2005

A highly specific labeling of a phosphate‐transporting protein from rat‐liver mitochondria was obtained with [203Hg]mersalyl under the following conditions: (a) labeling of mitoplasts, isolated by the French‐Press procedure; (b) washing of the isolated inner membranes with NaCl/octylglucoside.

The concentration‐dependent labeling of the protein with mersalyl showed a biphasic saturation which correlated well with the protection and inhibition of phosphate transport.

The molecular weight (Mr 31000) and the amount of labeled carrier protein (24 nmol/g mitochondrial protein) at 100 protection correlated well with published data from the literature.

Gel filtration of the labeled protein in the presence of non‐ionic detergents showed a molecular weight of at least twice of that obtained by gel electrophoresis in the presence of sodium dodecylsulfate.
Regulation of Ca2+ Efflux from Kidney and Liver Mitochondria by Unsaturated Fatty Acids and Na+ Ions
Authors: ROMAN Izabela , GMAJ Piotr , NOWICKA Cecylia , ANGIELSKI Stefan
Published: March 2005

The effects of fatty acids and monovalent cations on the Ca2+ efflux from isolated liver and kidney mitochondria were investigated by means of electrode techniques. It was shown that unsaturated fatty acids and saturated fatty acids of medium chain length (C12 and C14) induced a Ca2+ efflux from mitochondria which was not inhibited by ruthenium red, but was specifically inhibited by Na+ and Li+. The Ca2+‐releasing activity of unsaturated fatty acids did not correlate with their uncoupling activity. In kidney mitochondria a spontaneous, temperature‐dependent Ca2 efflux was observed which was inhibited either by albumin or by Na+. It is suggested that the net Ca2+ accumulation by mitochondria depends on the operation of independent pump and leak pathways. The pump is driven by the membrane potential and can be inhibited by ruthenium red, the leak depends on the presence of unsaturated fatty acids and is inhibited by Na+ and Li+. It is suggested that the unsaturated fatty acids produced by mitochondrial phospholipase A2 can be essential in the regulation of the Ca2+ retention in and the Ca2+ release from the mitochondria.
Studies on the Mechanism of the Rate‐Enhancing Effect of Heparin on the Thrombin‐Antithrombin III Reaction
Authors: HOLMER Erik , SÖDERSTRÖM Gunilla , ANDERSSON Lars‐Olov
Published: March 2005

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several mechanisms for this effect are possible. To study the problems commercial heparin was fractionated into one fraction of high anticoagulant activity and one of low anticoagulant activity by affinity chromatography on matrix‐bound antithrombin III. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed immunoelectrophoresis technique. As was to be expected, the high activity fraction was strongly bound to antithrombin III while the low activity fraction was weakly bound. In contrast, thrombin showed equal binding affinity for both heparin fractions. The ability of the two heparin fractions to catalyse the inhibition of thrombin by antithrombin III was determined and was found to be much greater for the high activity heparin fraction. A mechanism for the reaction between thrombin and antithrombin III in the presence of small amounts of heparin is suggested, whereby antithrombin III first binds heparin and this complex then inhibits thrombin by interaction with both the bound heparin and the antithrombin III.
Purification and Characterization of a Chymotrypsin‐Like Enzyme (Protease‐S) in Thermally Injured Rat Skin
Authors: MIYAKE Yoshimasha , ITO Toshiko
Published: March 2005

Protease‐S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G‐50, CM‐Sephadex (A‐50), Sephadex G‐75 gel filtration.

The optimum pH of this enzyme was 8.6–8.8, and the molecular weight determined by Sephadex G‐75 gel filtration was approximately 30000. This enzyme is active on the N‐acetyl‐L‐tyrosine ethyl ester, N‐succinyl‐L‐phenylalanine‐p‐nitroanilide (of chymotrypsin substrate) but not N‐tosyl‐L‐arginine methyl ester, N‐benzoyl‐L‐arginine‐p‐nitroanilide. Also, protease‐S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 μM), and N‐tosyl‐L‐phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin‐like proteases in mast cells and leukocyte granules. When protease‐S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.
Crystal Structure Analysis of the Tetragonal Crystal Form and Preliminary Molecular Model of Pig‐Heart Citrate Synthase
Authors: WIEGAND Georg , KUKLA Dietmar , SCHOLZE Henning , JONES T. Alwyn , HUBER Robert
Published: March 2005

The crystal structure of pig heart citrate synthase was analyzed at 0.35‐nm resolution. Chain tracing was possible and an initial molecular model constructed. The dimensions of the dimer molecule (located on a crystallographic diad) are 7.5 × 6.0 × 9.0 nm. The chain folding is characterized by the predominance of helices and the absence of sheet structure. The electron density accounts for 355 residues per monomer, so that about 80 residues must be disordered in the crystal. The disordered segment is probably N‐terminal. The ordered part consists of two closely associated domains, a large domain with 300 residues and a C‐terminal domain of 55 residues consisting of 3 (anti)parallel helices. The large domain is built from 12 helical segments, some of which are buried in the interior of the molecule. Inhibitor binding studies with citrate and CoA revealed citrate binding sites but showed no electron density for CoA. It is suggested that CoA binds to the disordered, flexible N‐terminal domain. Experiments of limited proteolysis with trypsin showed that under certain conditions a segment of Mr 9000 is cleaved off selectively. The remaining 35000‐Mr part is dimeric.
Identification of the Amino Acid Residue Modified in Bacillus stearothermophilus Alcohol Dehydrogenase by the NAD+ Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine
Authors: JECK Reinhard , WOENCKHAUS Christoph , HARRIS J. Ieuan , RUNSWICK Michael J.
Published: March 2005

4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl‐14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme‐coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site.

B. stearothermophilus alcohol dehydrogenase modified with 14C‐labelled coenzyme analogue and subsequently carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys‐38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys‐43 in yeast alcohol dehydrogenase and Cys‐46 in the horse liver enzyme but, unlike the latter two, Cys‐38 is not reactive towards iodoacetate in the native bacterial enzyme.
Affinity Labelling of Yeast and Liver Alcohol Dehydrogenases with the NAD Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine
Authors: WOENCKHAUS Christoph , JECK Reinhard , JÖRNVALL Hans
Published: March 2005

The NAD analogue 4‐(3‐bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo first order. The stoichiometry is one mole inactivator incorporated per mole enzyme subunit.

The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labelling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labelled on the essential cysteine residue 43.
The Complete Amino‐Acid Sequence of Histone H2B from the Mollusc Patella granatina
Authors: van HELDEN Paul D. , STRICKLAND Walter N. , BRANDT Wolf F. , von HOLT Claus
Published: March 2005

1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids.

2. In the carboxy‐terminal two thirds of the molecule there is a high degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions.

3. In the N‐terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.
Phase Behaviour of Cord Factor and Related Bacterial Glycolipid Toxins
Authors: DURAND Elisabeth , WELBY Michele , LANEELLE Gilbert , TOCANNE Jean‐François
Published: March 2005

C‐6 esters of methyl α‐D‐glucoside and C‐6, C‐6′ diesters of α,α′‐D‐trehalose with C18 and C32threo and erythro mycolic acids (from chemical source) and of C80‐erythro‐mycolic acid (from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well.

Throughout a monolayer study at the air‐water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction.

Compression isotherms of C32 esters suggested a transition between liquid‐expanded and liquid‐condensed states. Latent heats Qc and entropy changes ΔS associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular, intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32‐erythro‐mycoloyl)‐trehalose.

On the other hand, short chain C18 esters form fluid phases (t > 10°C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t= 20°C).

These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3‐sn‐phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes.
270‐MHz Nuclear‐Magnetic‐Resonance Study on Linear Gramicidin A in Dimethylsulfoxide
Authors: HEITZ Frédéric , SPACH Gérard , CARY Peter D. , CRANE‐ROBINSON Colyn
Published: March 2005

On the basis of the finding of two sets of coupling constants (8.8 Hz and 6.7 Hz) in the 270‐MHz proton magnetic resonance spectra of linear Gramicidin in dimethylsulfoxide and the comparison of its infrared spectrum with those of known conformations of alternating poly(γ‐benzyl‐D‐glutamate–γ‐benzyl‐L‐glutamate), it is proposed that the antibotic has an LD‐ribbon structure.
Dissociation and Association of Fumarase Subunits with Special Reference to the Formation of a Functional Tetramer
Authors: YAMATO Susumu , MURACHI Takashi
Published: March 2005

Swine heart fumarase is known to exist as a tetramer which can be dissociated with denaturing agents into inactive subunits. We have succeeded, for the first time, in reverting once‐dissociated subunits to form a functional tetramer, and studied the mechanism of the restoration of its activity.

Incubation of fumarase in a medium containing 50 mM sodium phosphate buffer, pH 7.3, 10 mM dithiothreitol, 0.1 mM EDTA · 2 Na, 10% glycerol, 200 mM KCl and 6 M urea at 25°C for 30 min resulted in dissociation of the tetrameric enzyme into monomeric subunits which were totally inactive and lost most α‐helical structure, with all sulfhydryl groups freely accessible to the medium. The denatured subunits could then be reassociated by dialysis at 4°C for 24 h against the same medium as above with omission of 6 M urea. The product was found to be an inactive dimer which could be transformed into an enzymatically active tetramer only after incubation at 25°C for 3 h. The final product was indistinguishable from the native fumarase in physical, chemical and enzymatic properties.

All the ingredients of the media were found to be necassary to achieve freely reversible and complete conversion of monomer to tetramer. An intermediate product, a dimer, showed fluorescence and circular dichroism spectra almost indistinguishable from those of a tetramer, but it was highly susceptible to tryptic digestion while the tetramer was strongly resistant. It is concluded that a small but very important conformational change associates with the conversion of dimers to tetramers, finalizing proper orientation of the active site residues.
NADPH‐Cytochrome c Reductase of Candida tropicalis Grown on Alkane
Authors: BERTRAND Jean Claude , BAZIN Hermine , ZACEK Marcelle , GILEWICZ Michèle , AZOULAY Edgard
Published: March 2005

During growth on n‐tetradecane Candida tropicalis synthesizes a NADPH‐cytochrome c reductase whose level is 4‐times higher than that of cells grown on glycerol and 20‐times higher than that of cells grown on glucose. An original method of solubilisation of this enzyme based on an osmotic shock is described. Studies of the purified enzyme showed that it is a flavoprotein having an apparent molecular weight of 67000 (± 1000) containing 1 mol each of FMN and FAD per mol of enzyme. Dichloroindophenol and ferricyanide are reduced by this enzyme. NADPH is reduced in the presence of menadione. The Michaelis constant for cytochrome c and NADPH are respectively equal to 4.55 μM and 29 μM. The maximal activity is obtained at a pH comprised between 7.6 and 8. It is influenced by ionic strength and inhibited by thiol inhibitors.

The NADPH‐cytochrome c reductase present in the cells grown on glycerol was isolated and purified. Its properties were found identical to those of the enzyme present in the cells grown on tetradecane, including its apparent molecular weight. However, its physiological function remains to be determined. Effectively we have shown that the cells grown on glycerol do not synthesize cytochrome P‐450 and the microsomal fraction prepared from these cells does not have any hydroxylase activity with dodecanoic acid as substrate.

This flavoprotein involved in the hydroxylation of the alkanes is a cytochrome c reductase, probably a cytochrome P‐450 reductase.
Photoreactions of Cytochrome b563 and f554 in Intact Spinach Chloroplasts: Regulation of Cyclic Electron Flow
Authors: BÖHME Herbert
Published: March 2005

In intact spinach chloroplasts cytochrome b563 and cytochrome f show an antagonistic redox behaviour in the extent and kinetics of light/dark‐induced absorbance changes. The extent of cytochrome b563 photoreduction appears to be influenced by the redox state of plastoquinone suggesting a regulation of cyclic electron flow at this level. Far‐red illumination induces a membrane state with altered redox reactions of both cytochromes at a more oxidized level.
Response of Specific Transfer‐Ribonucleic‐Acid Levels to Amino‐Acid Deprivation in Friend Leukemia Cells
Authors: WEISER Kristina , LITT Michael
Published: March 2005

In eukaryotes, the levels of specific tRNAs are closely correlated with the demands for their cognate amino acids in protein synthesis. To account for this phenomenon, we have proposed that the extent of aminoacylation of a given tRNA species in vivo controls the relative rate of synthesis or turnover of that species. Previously, we reported that Friend leukemia cells respond to histidine deprivation by increasing their relative level of tRNAHis by as much as two‐fold, with no change in the relative level of tRNALeu. In this paper, we show that deprivation of leucine or tryptophan also causes a specific increase in the relative level of tRNAs cognate to the deprived amino acid. At least in the case of tRNATrp, the increases in relative tRNA levels are preceded by extensive declines in the steady‐state extent of aminoacylation of the tRNA in vivo. We also find that different isoacceptors may respond differently to amino acid deprivation. These results suggest that decreased extents of aminoacylation of a given tRNA species in vivo cause increases in the relative rate of synthesis or decreases in the relative rate of degradation of that species.
Properties of Two Molecular Forms of β‐Glucuronidase from the Mollusc Littorina littorea L.
Authors: DIEZ Trinidad , CABEZAS José A.
Published: March 2005

The occurrence of two molecular forms, I and II, in the β‐glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355‐fold and 1262‐fold, respectively. Form I and II of β‐glucuronidase behave differently on DEAE‐cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4–4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p‐nitrophenyl β‐d‐glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G‐200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This β‐glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with β 1→3 linkages; it had practically no activity on certain glycosaminoglycans. Hg2+ and glucaro‐1,4‐lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.
Binding of Oligonucleotides to the Disk of Tobacco‐Mosaic‐Virus Protein
Authors: GRAHAM James , BUTLER P. Jonathan G.
Published: March 2005

The trinucleoside diphosphate A‐A‐G and the hexanucleotide fraction from a ribonuclease I digest of yeast RNA have been soaked into crystals of the disk aggregate of tobacco mosaic virus protein. At high concentrations these cause disruption of the crystal, probably by mimicking the normal nucleation of assembly.

At lower nucleotide concentrations the crystals remain intact and the differences caused by nucleotide binding have been studied by X‐ray diffraction. The most obvious change is an upwards movement of about 0.3 nm at the low‐radius end of the left radial helix in the protein with some stiffening of the helix so that it now extends visibly in from 4 nm to 3 nm radius. Similar shifts also occur in the right radial and left slewed helices.

A positive peak, which is tentatively identified with the bound oligonucleotide, is seen around 4 nm radius and below the right radial helix. The amino acid residues in possible contact with this feature are discussed.
Cobalt(III) Labeling of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: KALOGERAKOS Théodore , BLANQUET Sylvain , WALLER Jean‐Pierre
Published: March 2005

Native and trypsin‐modified methionyl‐tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP‐Co(II).

It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP‐Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site.

The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2‐mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.
Defect in the Split Proteins of 30‐S Ribosomal Subunits and Under‐Methylation of 16‐S Ribosomal RNA in a Polymamine‐Requiring Mutant of Escherichia coli Grown in the Absence of Polymines
Authors: IGARASHI Kazuei , KASHIWAGI Keiko , KISHIDA Kazuo , WATANABE Yasuhiro , KOGO Atsuko , HIROSE Seiyu
Published: March 2005

Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50‐S ribosomal subunits and reconstituted 30‐S particles containing different combinations of 23‐S core particles and 30‐S subunit split proteins obtained from a polyamine‐requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30‐S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine‐requiring mutant of E. coli grown in the absence of polyamines.

The methylation of 16‐S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23‐S RNA did not change significantly. The decrease in methylation of 16‐S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine.

The relationship between the decrease of polypeptide synthetic activity of 30‐S ribosomal subunits obtained from a polyamine‐requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16‐S RNA is discussed.
Interaction of Human Serum Albumin with Fatty Acids
Authors: SHANBHAG Vithaldas P. , JOHANSSON Göte
Published: March 2005

The interaction of human serum albumin with fatty acids has been determined using the method of affinity partitioning in aqueous biphasic systems containing dextran, poly(ethylene glycol) and esters of dicarboxylic acids with poly(ethylene glycol). The difference in the partition of albumin in phase systems with and without the poly(ethylene glycol)‐bound fatty acid group provides a measure of the interaction of fatty acids with the protein. The relative contribution of the polar and non‐polar interaction to the binding of fatty acids to albumin has been estimated by comparing the present data with that obtained earlier using poly(ethylene glycol)‐bound straight chain aliphatic hydrocarbons. In both cases, the aliphatic chain should contain a minimum of 8 carbon atoms to affect the partition of albumin and that the maximum effect is obtained with chains containing 16 carbon atoms. The effect of the polymer‐bound fatty acid group is higher than the corresponding hydrocarbon only when the number of carbon atoms in it exceeds 12. The relative effect of polymer‐bound 16‐carbon chains, with and without a carboxyl group in the terminal position is independent of pH in the range 5–9.
A DNA‐Unwinding Enzyme Induced in Bacteriophage‐T4‐Infected Escherichia coli Cells
Authors: KRELL Herbert , DÜRWALD Hildegard , HOFFMANN‐BERLING Hartmut
Published: March 2005

A single‐stranded DNA‐dependent ATP γ‐phosphohydrolase of Mr 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and Mg2+ ions it is capable of dissociating partially double‐stranded fd bacteriophage DNA into the single strands and that some 3000 enzyme copies are required to unwind the 6400‐nucleotides‐long DNA. Unwinding is inhibited by reducing the length of the single‐stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single‐stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double‐stranded DNA is not detectable by DNA binding assay.
Properties of Reconstituted Cholesterol 7α‐Hydroxylase System from Rat and Rabbit Liver Microsomes
Authors: HANSSON Ronnie , WIKVALL Kjell
Published: March 2005

7α‐Hydroxylation of cholesterol was studied in reconstituted systems containing cytochrome p‐450 and NADPH‐cytochrome P‐450 reductase from rat and rabbit liver microsomes. NADPH‐cytochrome P‐450 reductase was prepared with a biospecific affinity chromatographic method. Cytochrome P‐450 was prepared by solubilization with non‐ionic detergents and various chromatographic procedures. After the last purification step two fractions of cytochrome P‐450 from rat liver were analyzed for cholesterol 7α‐hydroxylase activity. One fraction had low or no detectable 7α‐hydroxylase activity but was able to catalyze 7α‐hydroxylation of taurodeoxycholic acid, and 11‐hydroxylation and 12‐hydroxylation of lauric acid. The other fraction catalyzed an efficient 7α‐hydroxylation of cholesterol as well as 7α‐hydroxylation of cholestanol. This fraction also catalyzed 12α‐hydroxylation and 26‐hydroxylation of 5β‐cholestane‐3α,7α‐diol, 7α‐hydroxylation of taurodeoxycholic acid and, 11‐hydroxylation and 12‐hydroxylation of lauric acid. Three cytochrome P‐450 fractions from rabbit liver, obtained after the final purification step, were analyzed for cholesterol 7α‐hydroxylase activity. Two fractions had no detectable 7α‐hydroxylase activity but were able to catalyze 12α‐hydroxylation and 25‐hydroxylation of 5β‐cholestane‐3α,7α‐diol, 25‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol and 11‐hydroxylation and 12‐hydroxylation of lauric acid. The third fraction catalyzed in addition 7α‐hydroxylation of cholesterol and cholestanol.

The cholesterol 7α‐hydroxylase activity showed an absolute requirement for cytochome P‐450 and NADPH‐cytochrome P‐450 reductase. Phospholipid had no significant effect on cholesterol 7α‐hydroxylation.
Translation of Alfalfa‐Mosaic‐Virus RNA 1 in the mRNA‐Dependent Translation System from Rabbit Reticulocyte Lysates
Authors: VAN TOL Ruud G. L. , VAN VLOTEN‐DOTING Lous
Published: March 2005

Translation of alfalfa mosaic virus (AMV) RNAs in the mRNA‐dependent rabbit reticulocyte cell‐free system was examined using different RNA concentrations. The pattern of products synthesized under the direction of AMV RNA 2, 3 and 4 was not or almost not influenced by their concentration. However, depending on the RNA 1 concentration either a very large protein of Mr 115000 or a mixture of two smaller proteins, Mr 58000 and 62000 respectively, was formed. These three proteins represent overlapping peptide chains with identical N‐termini. Addition of the cap analogue 7‐methylguanosine 5′‐monophosphate (m7GMP) or AMV RNA 3 stimulated the production of the 115000‐Mr protein at the expense of the 58000‐Mr and 62000‐Mr proteins. Both m7 GMP and RNA 3 probably reduce the active concentration of RNA 1 by competing for (a) cellular component(s) necessary for translation. These experimental results suggest that the rate of translation beyond the C termini of the 58000‐Mr and 62000‐Mr proteins is reduced or completely inhibited owing to the limited availability of the succeeding tRNA(s).
Protein Interactions in the Outer Membrane of Escherichia coli
Authors: PALVA E. Tapio
Published: March 2005

Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross‐linking with truly cleavable reagents and symmetrical two‐dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The major outer membrane proteins were shown to form cross‐linked complexes. These included multimers of λ receptor, protein I, II*, III and the free form of lipoprotein. Lipoprotein was also found to be cross‐linked to proteins II* and III. The identity of many of these complexes was verified using appropriate mutants missing the proteins in question. No new protein interactions were detected in the mutants even when three of the major proteins were missing. Proteins II*, III and the free form of lipoprotein could also be cross‐linked to the peptidoglycan layer of the cell wall.
Kinetic Characterization of Tissue‐Specific Isozymes of Octopine Dehydrogenase from Mantle Muscle and Brain of Sepia officinalis
Authors: STOREY Kenneth B. , STOREY Janet M.
Published: March 2005

Octopine dehydrogenase, the terminal enzyme of anaerobic glycolysis in the cuttlefish, Sepia officinalis, displays kinetically distinct tissue‐specific isozymic forms. An initial survey of octopine dehydrogenase from eleven tissues of Sepia revealed that only brain octopine dehydrogenase showed significant substrate inhibition by pyruvate. This property, which is characteristic of H‐type lactate dehydrogenase, was used as the basis for a study comparing and contrasting the kinetic properties of the mantle muscle and brain isozymes of octopine dehydrogenase. Compared to the mantle muscle enzyme, brain octopine dehydrogenase displayed: (a) a higher apparent affinity for the reactants of the reverse reaction (Km for octopine was 10‐fold lower than that of the muscle enzyme), (b) stronger substrate inhibition by both pyruvate and octopine, (c) greater inhibition by the product of the forward reaction, octopine, (d) an increased activity with the hypoxanthine derivative of NADH, and (e) an inhibition of enzyme activity in an incubate containing NAD+, pyruvate, and arginine (indicative of the formation of an inhibitory complex, enzyme‐pyruvate‐arginine‐NAD+). The kinetic properties of the mantle muscle and brain isozymes of octopine dehydrogenase and the differences between these two forms appear analogous to the well‐known properties of the M4 versus H4 isozymes of lactate dehydrogenase. Mantle muscle octopine dehydrogenase, with kinetic properties resembling the M form of lactate dehydrogenase, appears geared for the rapid synthesis of octopine under conditions of muscular work. Brain octopine dehydrogenase, like H‐type lactate dehydrogenase, displays properties which may poise the enzyme for a major role in the oxidation of octopine in vivo.
Effects of Spin‐Labeled Stearates on Duck Erythrocyte Adenylate Cyclase
Authors: TRAN Phuong L.
Published: March 2005

The effects of various spin‐labeled stearates on duck erythrocyte adenylate cyclase were investigated. Only 2‐(3‐carboxypropyl)‐4,4‐dimethyl‐2‐tridecyl‐3‐oxazolidinyloxyl caused an increase in adenylate cyclase activity. It increased the basal rate by about 50%, and the activities stimulated by isoproterenol and isoproterenol plus guanosine 5′‐[β,γ‐imido]triphosphate by 35%. Upon analysis of the width parameter Δ1 in the electron spin resonance spectra for both the basal enzyme activity and the stimulation obtained with effectors such as guanosine 5′‐[β,γ‐imido]triphosphate, isoproterenol, isoproterenol plus guanosine 5′‐[β,γ‐imido]triphosphate and NaF, a correlation of the changes of modification in adenylate cyclase activities was found. These findings suggest that the molecular environment of the enzyme has been modified.
On the Function of the Ribosomal Protein S1 in the Elongation Cycle of Bacterial Protein Synthesis
Authors: LINDE Richard , KHANH Nguyen QUOC , LIPECKY Rolf , GASSEN Hans Günter
Published: March 2005

The function of the ribosomal protein S1 in the elongation cycle of bacterial protein synthesis was examined. S1 was removed from 30‐S ribosomal subunits by adsorption of the subunits to a column of Sepharose 4B linked to anti‐S1 IgG, followed by elution of the 30S (−1) subunits with 0.8 M NH4Cl. The depleted 30‐S subunits showed, when combined with 50‐S subunits, a reduced activity in poly(U)‐dependent polyphenylalanine synthesis and in poly(A)‐stimulated polylysine synthesis. The ribosomes could be reactivated in these functions by the addition of stoichiometric amounts of S1. The association constants (K1) for the binary complex between 30‐S subunit and oligonucleotide, as measured by equilibrium dialysis, revealed no S1 effect. Whereas the activity of 30‐S subunits and 70‐S ribosomes in the coded binding of Phe‐tRNA was influenced by the chain length of the template and the presence of S1, neither effect was found in the oligo(A)‐directed binding of Lys‐tRNA. Reconstitution of the Phe‐tRNA binding capacity of depleted 30‐S and 70‐S particles by the addition of S1 was dependent on the chain length of the oligouridylate. The association constant (K2) for the binding of Phe‐tRNA to the 30‐S‐subunit · oligonucleotide complex was S1‐dependent with oligo(U) as template. It is proposed that S1, in analogy to its postulated function in initiation, binds to the phosphodiester backbone of the mRNA and adjusts the conformation of the codon to optimize double‐strand formation with the anticodon of a cgnate tRNA
An Apolipoprotein Homolog of Rat Apolipoprotein A‐IV in Human Plasma
Authors: BEISIEGEL Ulrike , UTERMANN Gerd
Published: March 2005

A protein with properties similar to apoprotein A‐IV of Rat high‐density lipoproteins has been isolated from the lipoprotein fraction (δ < 1.006 g/ml) of individual non‐fasting hypertriglyceridaemic subjects. The Mr of human apolipoprotein A‐IV determined by polyacrylamide‐gel electrophoresis in sodium dodecylsulfate and by column chromatography in 6 M guanidine hydrochloride is 46000. The amino acid composition is significantly different from any previously characterised human apolipoprotein but resembles that of rat apolipoprotein A‐IV. The N‐terminal amino acid of human and rat apolipoprotein A‐IV is glutamate or glutamine. The pI of the major human A‐IV band in 6 M urea is ∼ 5.15. Immunochemically apolipoprotein A‐IV is not identical with any of the well known human apolipoproteins. Human apolipoprotein A‐IV is present in all fractions of δ < 1.006 g/ml isolated by a single ultracentrifugal spin from non‐fasting subjects, but is lost from lipoproteins of δ < 1.006 g/ml upon recentrifugation.

Immunoelectrophoretic studies show that most of human apolipoprotein A‐IV is present in the fraction of δ > 1.21 g/ml and is unassociated with the major lipoprotein fractions in serum. Charge‐shift electrophoresis however clearly demonstrates the hydrophobic character of human apolipoprotein A‐IV. There is evidence that apolipoprotein A‐IV is a constituent of chylomicrons that is removed from the particle surface when chylomicrons enter the plasma compartment.
The Role of the Reactive Disulfide Bond in the Interaction of Cholera‐Toxin Functional Regions
Authors: TOMASI Maurizio , BATTISTINI Angela , ARACO Antonio , RODA L. Giorgio , D'AGNOLO Giuliano
Published: March 2005

The chemical reactivity of disulfide bonds towards reducing agents, in the absence of denaturing conditions, in cholera toxin has been studied. Treatment of the toxin with dithiothreitol or other mercaptans gave selective reduction of one of the six disulfide bonds of the protein. This reactive disulfide links two distinct functional regions of the toxin, fragment α, which activates adenylate cyclase, and fragment γβ5, which recognizes the cell surface receptors. Upon reduction, the two fragments remain bound together and the secondary structure of the protein is retained. The two functional regions have been separated and purified only by methods based on charge differences. When mixed together, purified α and purified γβ5 fragments spontaneously and rapidly re‐form the disulfide bond. However, reduction of the disulfide bond is an absolute requirement for freeing the catalytic site of the α functional region. Thus, while other non‐covalent binding regions are involved in maintaining cholera toxin molecular structure, the reactive disulfide bond may play a role in the mechanism of cell intoxication.
Immunological Cross‐Reactivity between Electric‐Eel Acetylcholinesterase and Rat‐Tail‐Tendon Collagen
Authors: ANGLISTER Lili , TARRAB‐HAZDAI Rebecca , FUCHS Sara , SILMAN Israel
Published: March 2005

Immunological cross‐reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels.

Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed‐type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14‐S + 18‐S molecular forms). However, when the glubular 11‐S enzyme was tested, almost no cross‐reactivity was obtained. Similarly, guinea pigs immunized with 14‐S + 18‐S preparation exhibited skin sensitization to rat tail tendon collagen.

Using a radioimmunoassay, it was observed that 125I‐labeled 14‐S + 18‐S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I‐labeled 11‐S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay.

The experiments suggest that 14‐S + 18‐S acetylcholinesterase, but not 11‐S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.
Hydrophobic Interactions of Cytochrome c Oxidase
Authors: NAGASAWA Toru , NAGASAWA‐FUJIMORI Haruko , HEINRICH Peter C.
Published: March 2005

The binding of rat liver cytochrome c oxidase to phenyl‐Sepharose and various alkyl and ω‐aminoalkyl agarose gels has been studied. Deoxycholate‐solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, ω‐aminohexyl, ω‐aminooctyl agarose as well as to phenyl‐Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl‐Sepharose was devoid of NADH (NADPH)‐acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40000, 23200, 21500, 14500, 12600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross‐react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl‐Sepharose or phenyl‐Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed.
Ribosome‐Protected Fragments from Sindbis 42‐S and 26‐S RNAs
Authors: CANCEDDA Ranieri , SHATKIN Aaron J.
Published: March 2005

Sindbis virus 42‐S and 26‐S RNAs labeled with 32P were purified from infected chick embryo fibroblasts. The RNAs were incubated in the presence of a wheat germ cell‐free translating system under conditions that yielded 40‐S and 80‐S initiation complexes. After digestion with RNase A, ribosome‐protected fragments were isolated by polyacrylamide gel electrophoresis and compared with respect to number, size, cap content and oligonucleotide composition. The two RNA species yielded several fragments of chain length about 35–40 nucleotides from 80‐S complexes and up to 60–65 nucleotides from 40‐S complexes. The 5′‐terminal capped sequence, m7GpppA‐U‐G that is present in both Sindbis virus RNAs, was not retained in any of the ribosome‐protected fragments. Fingerprint analyses indicated that the fragments derived from 40‐S and 80‐S initiation complexes of each species of RNA were overlapping, but the fragments from 42‐S and 26‐S RNAs were unrelated. The complexity of the fingerprints were consistent with protection of a single, different initiation site in each Sindbis virus RNA.
The ‘DNA‐Membrane Complex’ of Escherichia coli B/r
Authors: ROBERTSON William , WATKINS David
Published: March 2005

The composition and properties of the ‘DNA‐membrane complex’ of Escherichia coli B/r have been investigated. The ‘complexes’ contain most of the DNA and membrane of the cells, and about 50% and 25% of the RNA and protein respectively. The properties of DNA synthesized by the ‘complexes’ are described and the process is concluded to be largely mediated through polymerase I.

Nascent DNA synthesized by the ‘DNA‐membrane complexes’ was of two main classes, one of molecular weight around 600000–800000 and the other of higher molecular weight. Polynucleotide ligase activity was not detectable. The onset of synthesis coincided with the dissociation of at least 70% of the genome DNA and all of the nascent DNA from the ‘complexes’ and was concomitant with the action of a nuclease on parental DNA. This nuclease activity was not ATP‐dependent.