bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of DNA conformation on the transcription of mitochondrial DNA
Authors: BARAT‐GUERIDE Monique , DUFRESNE Catherine , RICKWOOD David
Published: March 2005

The importance of the conformation of the DNA template for in vitro transcription experiments was investigated using a mitochondrial RNA polymerase preparation from Xenopus laevis oocyte mitochondria. A premature termination transcription assay has been devised and the levels of the formation of transcriptional complexes have been identified using agarose gel electrophoresis. It was found that accurate initiation of transcription from in vivo promoter sequences was enhanced by supercoiling and that the two mitochondrial DNA‐binding proteins, previously characterized by us, inhibited the transcription of DNA as gauged by these assays.
The genes for human alcohol dehydrogenases β1 and β2 differ by only one nucleotide
Authors: MATSUO Yoshinori , YOKOYAMA Ruth , YOKOYAMA Shozo
Published: March 2005

Allelic differences at the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase loci may have an important role in an individual's alcohol sensitivity. We have cloned and sequenced all nine exons of an ADH22 allele which codes for an ‘atypical’ ADH, ADHβ2. Our sequence data shows that the histidine at residue 47 of ADHβ2 is encoded by CAC. Surprisingly, no silent substitution was found between the coding regions of ADH21 [Duester, G., Smith, M., Bilanchone, V. & Hatfield, G. W. (1986) J. Biol. Chem. 261, 2027–2033.] and ADH22 alleles over the 1122 nucleotide sites.
Direct and energy‐transfer photolabelling of brain muscarinic acetylcholine receptors
Authors: ILIEN Brigitte , HIRTH Christian
Published: March 2005

Efficient photolabelling of muscarinic acetylcholine receptors was obtained using either two aryldiazonium salts or an azido derivative. These probes did not discriminate between muscarinic binding subtypes or affinity states and became irreversibly bound to the receptor sites, in an entirely atropine‐protectable manner, upon ultraviolet irradiation. The extent of labelling was dependent both on probe concentration and on time of irradiation and reached up to 80% of the receptor population, under optimal alkylating conditions.

In contrast to the azido derivative, both diazonium salts behave as potent irreversible labels of muscarinic receptors, provided energy‐transfer photolabelling conditions were followed. Such an indirect activation of diazonium ligands, through an energy transfer from photoexcited tryptophan residues, has been previously found to increase the site‐specificity and the rate of labelling of other acetylcholine binding proteins. Analogies in the photolabelling process of acetylcholinesterase or of nicotinic and muscarinic receptors by the two diazonium salts are discussed.

Altogether, these findings suggest that these new probes may be promising tools to investigate the location and the topography of the agonist‐antagonist binding domain on purified muscarinic receptors, through amino acid and/or sequence analyses of radioactive, photolabelled residues.
Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water‐in‐oil microemulsion system
Authors: OLDFIELD Christopher , FREEDMAN Robert B.
Published: March 2005

Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water‐in‐oil microemulsion, which consists of discrete nanometer‐diameter water droplets dispersed in a continuous water‐immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface.

Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule.

Studies are presented on the enzyme‐catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations.

The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis‐Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.
Iron metabolism of Escherichia coli studied by Mössbauer spectroscopy and biochemical methods
Authors: MATZANKE Berthold F. , MÜLLER Gertraud I. , BILL Eckhard , TRAUTWEIN Alfred X.
Published: March 2005

To date it has barely been recognized that the nature of about 75% of the Escherichia coli iron pool is unknown. Here we report the isolation of two iron species representing major components of iron metabolism in various growth states of E. coli. In vivo Mössbauer spectroscopy was applied to obtain information on the intracellular distribution pattern of iron in E. coli K12 W3110. Only two types of iron could be detected in the cell spectra: hexacoordinated Fe2+ and Fe3+ high‐spin complexes. Other iron‐requiring compounds are at least one order of magnitude less abundant in E. coli. The Mössbauer parameters of these complexes fit neither cytochromes nor iron‐sulfur proteins nor ferric holo‐bacterioferritin. They are sensitive to metabolic changes and inhibitors. The ratio of Fe/subunit. Fe2+/Fe3+ interconversion, chromatographic and electrophoretic data exclude bacterioferritin as the main iron metabolite in E. coli. Bacterioferritin can be observed only at very high ferric ion concentrations in the medium. The 55Fe fluorograms of both cytoplasmic and membrane fractions exhibit two exclusive bands with apparent molecular masses of 17 and 15 kDa, respectively. The two bands comprised 70% of the applied radioactivity. In gel filtration the main iron peak elutes at 155 kDa yielding two bands with apparent molecular masses of 17 and 15 kDa on SDS/PAGE. We therefore conclude that the iron species form a protein with an apparent molecular mass of 155 kDa containing 17‐kDa and 15‐kDa subunits. The iron content of the protein is 44 μg Fe/mg protein which corresponds to approximately 13 iron ions/subunit. No iron protein exhibiting the observed features has been described so far. Additional Mössbauer experiments suggest that these novel iron proteins are not restricted to E. coli but that similar components are detectable in several bacterial and fungal systems, thus pointing to a general occurrence.
Reduced temperature does not prevent transport of lysosomal integral membrane proteins from endoplasmic reticulum and through the Golgi system to lysosomes
Authors: MORALES Pilar García , BARRIOCANAL Javier G. , SANDOVAL Ignacio V.
Published: March 2005

The effect of low temperature on the transport of three lysosomal integral membrane proteins (I, II and III) from endoplasmic reticulum to lysosomes has been studied in normal rat kidney cells. At 15°C and 18°C, though slowly, the proteins could leave the endoplasmic reticulum, move through the Golgi system from the cis to the trans side, and accumulate in lysosomes. Transport of these proteins at low temperature occurred slower than at 37°C. Both at low temperature and 37°C, the proteins were transported between the endoplasmic reticulum and Golgi (III > I and II) and from Golgi to lysosomes (II > III > > I) with different rates.
The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase
Authors: BEECKMANS Sonia , VAN DRIESSCHE Edilbert , KANAREK Louis
Published: March 2005

Evidence is growing that the citric acid cycle, like many other metabolic pathways, might exist in vivo as a more or less tightly organized multi‐enzyme cluster. The term ‘metabolon’ [Robinson, J. B. & Srere, P. A. (1985) J. Biol. Chem. 260, 10800–10805] was recently introduced to describe such a complex of sequential metabolic enzymes.

We adopted the technique of affinity electrophoresis for the study of interactions between the cycle enzymes fumarase and malate dehydrogenase. This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J. Biochem. 117, 527–535], one of which is the fact that the interaction can be directly visualized. The observed association is specific since both metabolically unrelated proteins and the cytoplasmic isoenzyme of malate dehydrogenase do not interact with fumarase.

Several metabolites (citrate, isocitrate, 2‐oxoglutarate, succinate, fumarate, malate, oxaloacetate, Pi, AMP, ADP, NAD+, NADH) were found not to affect the association between fumarase and mitochondrial malate dehydrogenase. Both ATP, Mg2+‐ATP and GTP disrupt the association when they are present at 1 mM concentrations. Lower non‐physiological ATP concentrations do not, however, disturb the interaction. The presence of 1 mM ADP was found to abolish the disrupting effect of 1 mM ATP. The latter findings are suggestive of an interruption of the citric acid cycle at the level of fumarase under conditions of high energy load (i.e. high ATP/ADP ratios)
Nucleotide sequence of cDNA coding for saporin‐6, a type‐1 ribosome‐inactivating protein from Saponaria officinalis
Authors: BENATTI Luca , SACCARDO Maria Beatrice , DANI Maria , NITTI Gianpaolo , SASSANO Marica , LORENZETTI Rolando , LAPPI Douglas A. , SORIA Marco
Published: March 2005

We have isolated and sequenced partial cDNA clones that encode SO‐6, a ribosome‐inactivating protein from Saponaria officinalis. A cDNA library was constructed from the leaves of this plant and screened with synthetic oligonucleotide probes representing various portions of the protein. The deduced amino acid sequence shows the signal peptide and a coding region virtually accounting for the entire amino acid sequence of SO‐6. The sequence reveals regions of similarity to other ribosome‐inactivating proteins, especially in a region of the molecule where critical amino acid residues might participate in the active site.
Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides
Authors: SCHNEIDER Karl‐Heinz , GIFFHORN Friedrich
Published: March 2005

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D‐glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150‐fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q‐Sepharose and phenyl‐Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47200 as calculated from its Stokes' radius (rs= 2.76 nm) and sedimentation coefficient (s20.w= 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D‐glucitol and D‐mannitol to D‐fructose, as well as D‐arabinitol to D‐ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis‐Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D‐glucitol, and for D‐glucitol (31.8 mM), D‐mannitol (0.29 mM) and D‐arabinitol (1.8 mM), respectively. The KM value for D‐fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D‐mannitol, D‐glucitol and D‐arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D‐mannitol, an enzymatic assay for D‐mannitol was elaborated.
Purification and characterization of an acetyl‐CoA hydrolase from Saccharomyces cerevisiae
Authors: LEE Fang‐Jen S. , LIN Lee‐Wen , SMITH John A.
Published: March 2005

Acetyl‐CoA hydrolase, which hydrolyzes acetyl‐CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2‐terminally blocked. The enzyme was purified 1080‐fold to apparent homogeneity by successive purification steps using DEAE‐Sepharose, gel filtration and hydroxylapatite. The molecular mass of the native yeast acetyl‐CoA hydrolase was estimated to be 64 ± 5 kDa by gel‐filtration chromatography. SDS/PAGE analysis revealed that the denatured molecular mass was 65 ± 2 kDa and together with that for the native enzyme indicates that yeast acetyl‐CoA hydrolase was monomeric. The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8. Several acyl‐CoA derivatives of varying chain length were tested as substrates for yeast acetyl‐CoA hydrolase. Although acetyl‐CoA hydrolase was relatively specific for acetyl‐CoA, longer acyl‐chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl‐CoA. Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor. The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine‐modifying reagent.
Reactivity of the P‐site‐bound donor in ribosomal peptide‐bond formation
Authors: SYNETOS Dennis , COUTSOGEORGOPOULOS Charalambos
Published: March 2005

The puromycin reaction, catalyzed by the ribosomal peptidyltransferase, has been carried out so as to make the definition of two distinct parameters of this reaction possible. These are (a) the final degree of the reaction which gives the proportion of peptidyl (P)‐site binding of the donor and (b) the reactivity of the donor substrate expressed as an apparent rate constant (kobs). This kobs varies with the concentration of puromycin; the maximal value (k3) of the kobs, at saturating concentrations of puromycin, gives the reactivity of the donor independently of the concentrations of both the donor and puromycin. k3 is also a measure of the activity of peptidyltransferase expressed as its catalytic rate constant (kcat).

If we assume that the puromycin‐reactive donor is bound at the ribosomal P site, we observe the following, depending on the conditions of the experiment: the proportion of P‐site binding of the donor substrates AcPhe‐tRNA or fMet‐tRNA can be the same and close to 100%, while there is a tenfold increase in the reactivity of the donor (k3= 0.8 min−1 versus 8.3 min−1). On the other hand there are conditions, under which the proportion of P‐site binding increases from 30% to 100% while k3 remains low and equal to 0.8 min−1. Using the puromycin reaction it was also found that an increase of Mg2+ from 10 mM to 20 mM reduces the reactivity of the donor and, hence, the activity of peptidyltransferase, provided that this change in Mg2+ occurs during the binding of the donor but not when it occurs during peptide bond formation per se. The fact that the donor substrate may exist in various states of reactivity in this cell‐free system raises the possibility that the rate of peptide bond formation may not be uniform during protein synthesis.
Nuclear‐magnetic‐resonance investigation of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis
Authors: McGOURTY Jacqueline L. , LA MAR Gerd N. , SMITH Kevin M. , ASCOLI Franca , CHIANCONE Emilia , PANDEY Ravindra K. , SINGH Jai P.
Published: March 2005

The proton nuclear‐magnetic‐resonance spectra of the cyano‐met complexes of the cooperative dimeric and tetrameric hemoglobins from the mollusk Scapharca inaequivalvis have been investigated and compared to those of other structurally characterized oxygen binding hemoproteins. For these proteins, cooperativity is displayed even in the homodimer and preliminary X‐ray structural data reveal an unusual back‐to‐front assembly with intersubunit contacts involving the EF helices [Royer, W. E., Love, W. E. & Fenderson, F. F. (1985) Nature (Lond.) 316, 277–280]. The pattern of hyperfine shifts is very similar for the dimer and tetramer chains, but distinctly different from those of previously characterized low‐spin, ferric heme proteins. Individual heme resonances are identified by reconstituting the protein with specifically deuterated hemes. While the axial interactions involving the proximal and distal histidines are very similar to that in myoglobins and other hemoglobins, both the heme contact shift pattern and the amino acid dipolar shift pattern reflect a significantly reduced asymmetry. The decreased spread of the non‐cordinated amino acid signals is interpreted in terms of a rotation of the magnetic axes relative to those in myoglobin or other hemoglobins, rather than a change in the magnetic anisotropy. The decreased spread of the heme methyl contact shifts supports this conclusion and is consistent with an orientation of the proximal histidine with the imidazole ring rotated by about 30–40° relative to that in other structurally characterized proteins. Although resonances associated with a complex pattern of alternate heme orientations can be detected immediately after reconstitution of the protein, the isolated protein was found to exhibit insignificant equilibrium heme rotational disorder.
Methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum (strain Marburg)
Authors: ELLERMANN Joachim , ROSPERT Sabine , THAUER Rudolf K. , BOKRANZ Martin , KLEIN Albrecht , VOGES Markus , BERKESSEL Albrecht
Published: March 2005

Methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum (strain Marburg) was purified to a stage where, besides the α, β and γ subunits, no additional polypeptides were detectable in the preparation. Under appropriate conditions the enzyme was found to catalyze the reduction of methyl‐CoM with 7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP) to CH4 at a specific rate of 2.5 μmol · min−1· mg protein−1. This finding contradicts a recent report that methyl‐CoM reductase is only active when some contaminating proteins are present.

The two polypeptides encoded by the open reading frames ORF1 and ORF2 of the methyl‐CoM reductase transcription unit did not co‐purify with the α, β and γ subunits. They were neither required nor did they stimulate the activity under the assay conditions.

3‐Bromopropanesulfonate (apparent Ki= 0.05 μM) and 2‐azidoethanesulfonate (apparent Ki= 1 μM) were found to be two new competitive inhibitors of methyl‐CoM reductase. Both inhibitors were considerably more effective than the “classical” 2‐bromoethanesulfonate (apparent Ki= 4 μM).
γ‐Glutamylcyclotransferase: inhibition by d‐β‐aminoglutaryl‐l‐alanine and analysis of the solvent kinetic isotope effect
Authors: YORK Michael J. , CROSSLEY Maxwell J. , HYSLOP Serena J. , FISHER Megan L. , KUCHEL Philip W.
Published: March 2005

Spin‐echo NMR spectroscopy was used to record the cleavage of a γ‐glutamyl–amino‐acid by (5‐L‐glutamyl)‐L‐amino‐acid 5‐glutamyltransferase (cyclizing) (γ‐glutamylcyclotransferase) in human erythrocyte hemolysates. The Michaelis‐Menten steady‐state kinetic parameters were obtained by fitting the integrated Michaelis‐Menten equation to the reaction time curves. The product, L‐5‐oxoproline, was shown to be an inhibitor of the reaction. The active site of the enzyme was probed by studies of the inhibition by D‐ and L‐β‐aminoglutaryl‐L‐alanine which are the β‐amino‐acid isomers of D‐ and L‐γ‐glutamyl‐L‐alanine (the latter being a natural substrate of the enzyme); the D‐isomer was the more potent inhibitor (Ki= 0.30 ± 0.02 mmol/l water). When the alanyl α‐carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D‐β‐aminoglutaryl‐L‐alaninol) the potency of inhibition was reduced. The previously reported kinetic isotope effect of solvent 2H2O on the enzyme‐catalyzed reaction has been further studied using a proton inventory. We propose that the solvent kinetic isotope effect is due to an intramolecular proton transfer between the glutamyl amino group and the peptide bond nitrogen.
Alterations in plasma lipoproteins and apolipoproteins associated with estrogen‐induced hyperlipidemia in the laying hen
Authors: HERMIER Dominique , FORGEZ Patricia , WILLIAMS John , CHAPMAN M. John
Published: March 2005

The laying hen represents a physiological model in which the mechanisms of action of estrogens on lipid transport can be evaluated. The plasma lipoproteins in the laying hen were subfractionated into discrete particle species by isopycnic density gradient ultracentrifugation and the physicochemical properties and apolipoprotein contents of individual subfractions evaluated. The qualitative and quantitative aspects of this estrogen‐specific profile were then compared to those of the immature chicken. As observed earlier, estrogens induced dramatic elevation in very‐low‐density lipoproteins (VLDL) (up to 900 mg/dl). Indeed, triglyceride‐rich lipoproteins with densities up to 1.035 g/ml, i.e. VLDL and their remnants, behaved as a continuum which displayed little variation in size (20.5–21 nm), electrophoretic mobility (β‐like) and apolipoprotein content;apo B‐100 (540 kDa) predominated while apo A‐I (27 kDa), apo VLDL‐II (19 kDa) and an apo‐C‐like protein (13 kDa) were present as minor components. The typical high‐density lipoproteins (HDL) in the immature chicken were replaced by a lipoprotein population whose physicochemical properties were quite distinct. Thus these particles were distributed as a single, asymmetric peak over the density range 1.030–1.158 g/ml, a wide interval which overlapped that of apo‐B‐rich particles at its lower limit. The ρ 1.030–1.158 g/ml lipoproteins were present at concentrations (ρ 200 mg dl) some twofold to threefold lower than those of HDL in immature birds. Furthermore, they displayed physical and chemical properties in common with both low‐density lipoproteins (LDL) and HDL and were LDL‐like in exhibiting β mobility but HDL‐like in size (9–15 nm diameter). Their protein moiety was also HDL‐like in its predominant content of apo A‐I; small amounts of apo VLDL‐II and the apo‐C‐like protein were also detected. Substantial amounts of lipid were found at ρ > 1.195 g/ml: such substances are absent in the immature chicken and may reflect the presence of vitellogenins. The hyperestrogenic state in the laying hen is therefore associated with major modifications in lipoprotein and apolipoprotein profile. Such modifications may be of relevance to clinical disorders involving estrogen‐induced hyperlipidemia.
Water in the active cavity of copper/zinc superoxide dismutase
Authors: BANCI Lucia , BERTINI Ivano , HALLEWELL Robert A. , LUCHINAT Claudio , VIEZZOLI Maria Silvia
Published: March 2005

Water 1H‐nuclear‐magnetic‐relaxation‐dispersion (NMRD) measurements of solutions of several copper/zinc superoxide dismutase isoenzymes as well as mutants of the human isoenzyme have been performed in order to monitor the presence of exchangeable water at the copper(II) center. The results have been compared with other spectroscopic features of the various derivatives and with their catalytic efficiency. The decrease in the amount of water in the first coordination sphere, detected through NMRD, parallels, in most cases, the increase in the tetragonal nature of the copper ion. On the other hand, the catalytic activity seems unrelated to the presence of water. Most strikingly, the Ile137 mutant of the human isoenzyme, ∼90% active, has no water in the copper coordination sphere; this is taken as evidence that the electron transfer is not a water‐mediated process. The variation in the pH dependence of NMRD data between the wild‐type enzyme and the human Ile137 mutant has been found to parallel the variation in the pH dependence of activity.
Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca2+‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP
Authors: GIRARDET Jean‐Luc , DUPONT Yves , LACAPERE Jean‐Jacques
Published: March 2005

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+‐ATPase. Three classes of Tb3+‐binding sites have been found: a first class of low‐affinity (Kd= 10 μM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (<0.1 μM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+‐ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP.

Substitution of H2O by D2O shows that Tb‐FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+‐ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site.

Addition of calcium quenches the fluorescence signal of the terbium‐FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2→ E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1↔ E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high‐affinity calcium sites are located very close to the Tb‐FTP complex bound to the nucleotide site.
Molecular oxygen and the state of geometric isomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts
Authors: BEYER Peter , MAYER Matthias , KLEINIG Hans
Published: March 2005

The membrane‐bound carotenogenic reaction sequence in daffodil chromoplasts can be subdivided in vitro into three reaction segments by varying the incubation parameters O2 and light. In the first segment, 15‐cis‐phytoene is desaturated to 15‐cis‐ζ‐carotene (trans elimination of hydrogen) in the dark and in the presence of O2 as an essential cofactor. A photoisomerization of the 15‐cis double bond of the accumulated ζ‐carotene to trans is the prerequisite for the function of the second segment, the desaturation to 7,9,9′,7′‐tetra‐cis‐lycopene (prolycopene, cis elimination of hydrogen). The role of O2 as an electron acceptor is discussed and evidence for an oxidoreductase acting as a redox mediator between the desaturase (forming the polyene chromophore) and O2 is presented. A certain analogy to the desaturation of stearoyl‐carrier protein, which also occurs in plastids, is proposed. The third segment, the cyclization of prolycopene, is active only in the absence of O2 and involves additional cis‐trans isomerization reactions.
13C‐NMR study of autotrophic CO2 fixation in Thermoproteus neutrophilus
Authors: SCHÄFER Sigrid , GÖTZ Martina , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg
Published: March 2005

The pathway of autotrophic CO2 fixation has been investigated in the extremely thermophilic sulfur‐respiring anaerobic archaebacterium Thermoproteus neutrophilus. [1,4‐13C2]Succinate was used as a tracer since this compound was incorporated in small amounts virtually into all cell compounds without affecting the organism's ability to synthesize all cell constituents from CO2. Three representative amino acids, glutamate, aspartate and alanine were isolated from cells after growth for several generations in the presence of [1,4‐13C2]succinate and their labelling patterns were determined by 13C NMR spectroscopy.

The data is consistent with CO2 fixation by a reductive citric acid cycle, as proposed earlier for the green sulfur bacterium Chlorobium limicola, the sulfate‐reducing Desulfobacter hydrogenophilus and the microaerophilic Knallgasbacterium Hydrogenobacter thermophilus. The presence of a reductive citric acid cycle in archaebacteria indicates that this CO2 fixation mechanism which is an alternative to the Calvin cycle is present in many anaerobic or facultative anaerobic microorganisms.
Tightly bound DNA‐protein complexes representing stable attachment sites of large DNA loops to components of the matrix
Authors: PATRIOTIS Christos , DJONDJUROV Lalio
Published: March 2005

This study describes tightly bound DNA‐protein complexes in DNA of matrices isolated from Friend erythroleukemia cells. When after radio‐iodination of the associated proteins, such DNA is electrophoresed on agarose and the gel is subsequently subjected to autoradiography, the protein components of three or four complexes are visualized. Their two‐dimentional electrophoretic analysis revealed that each possesses a simple but specific polypeptide composition, including a set of five non‐histone proteins, characteristic for the matrix, and the core histones H3 and H4. Since the polypeptides dissociate from DNA by treatment with SDS, it is suggested that the linkage is not covalent. Reassociation and hybridization analysis of the DNA of the complexes indicated that it is enriched in highly repetitive, satellite sequences. The latter were found to be, to a great extent, similar to sequences localized at the base of large, dehistonized DNA loops obtained by high‐salt extraction of isolated nuclei. Further experiments emphasized the complete conservation of this type of attachment throughout erythroid differentiation of Friend cells. It is proposed that the complexes represent attachment sites of basic, 30–100‐kbp loop units of DNA.
Effects of the cellular p53 protein on Simian‐virus‐40‐T‐antigen‐catalyzed DNA unwinding in vitro
Authors: KIENZLE Hildegard , BAACK Martina , KNIPPERS Rolf
Published: March 2005

It is known that large T antigen, the regulatory protein encoded by Simian virus 40 (SV40), forms tight complexes with the cellular p53 protein in SV40‐transformed rodent cells. Using immunoaffinity procedures we have purified large T antigen and, in separate experiments, the cellular p53 protein. The two proteins formed complexes in vitro which bound well to double‐stranded DNA fragments although in a sequence‐unspecific manner. Free, uncomplexed T antigen readily converted double‐stranded DNA into a single‐stranded form whereas in‐vitro‐formed p53–T‐antigen complexes were inactive in this reaction. We conclude that one function of p53 in SV40‐transformed mouse cells could be the inhibition of the replication initiating activity of T antigen.
Modulatory activity of 9‐hydroxy‐ and 9‐hydroperoxy‐octadecadienoic acid towards reactive oxygen species from guinea‐pig pulmonary macrophages
Authors: ENGELS Ferdi , HENRICKS Paul A. J. , van der VLIET Helen , NIJKAMP Frans P.
Published: March 2005

As guinea‐pig pulmonary macrophages (PM) synthesize the linoleic acid metabolite 9‐hydroxy‐octadecadienoic acid (9‐OH‐Lin) under non‐stimulated conditions in relatively large quantities, we investigated whether this product has an effect on the macrophage's own phagocytic cell function. 9‐OH‐Lin, and also its hydroperoxy precursor 9‐hydroperoxy‐octadecadienoic acid (9‐OOH‐Lin), influenced the generation of PM chemiluminescence, a measure of the production of reactive oxygen species. The generation of lucigenin‐enhanced chemiluminescence by stimulated and non‐stimulated PM was inhibited concentration‐dependently. Inhibition was observed at concentrations as low as 10 nM. Since 9‐OH‐Lin and 9‐OOH‐Lin also inhibited the generation of chemiluminescence by a cell‐free enzyme system, i.e. xanthine/xanthine oxidase, the inhibitory effects might represent a scavenging activity towards reactive oxygen species. 9‐OH‐Lin and 9‐OOH‐Lin did not influence other phagocytic cell functions, e.g. PM phagocytic capacity, the aggregatory response to the calcium ionophore A23187, or the release of lysosomal enzymes. The effects of 9‐OH‐Lin and 9‐OOH‐Lin could be ascribed to the hydroxy and hydroxyperoxy moiety, respectively, as evidenced by lack of effect of the native fatty acid linoleic acid. We conclude that the formation of 9‐OH‐Lin and 9‐OOH‐Lin by PM may represent a regulatory mechanism towards the cell's own activity by modulating reactive oxygen species.
The effect of aqueous methanol cryosolvents on the heat‐ and cold‐induced denaturation of lactate dehydrogenase
Authors: HATLEY Ross H. M. , FRANKS Felix
Published: March 2005

The effect on the low‐temperature‐induced denaturation temperature of various concentrations of methanol has been studied for lactate dehydrogenase. The results have been compared to similiar data for the thermal denaturation temperature. Extrapolations of the low‐temperature data show that, in a physiological buffer in the absence of methanol, the cold denaturation temperature would be – 30°C. Data obtained with high concentrations of methanol indicate that residues are exposed to a similar degree upon either heat‐ or cold‐induced denaturation. Aggregation does not occur in the cold‐denatured protein. Cold‐induced denaturation is fully reversible at a protein concentration of 250 μg/ml. The spectra of the two denatured forms are similar.
Analysis of the cDNA for phospholipase A2 from honeybee venom glands
Authors: KUCHLER Karl , GMACHL Michael , SIPPL Manfred J. , KREIL Günther
Published: March 2005

A cDNA expression library was constructed from worker bee venom glands and screened with an antibody against phospho lipase A2. The nucleotide sequence of a positive clone with the largest insert showed an open reading frame that codes for part of the signal peptide, the pro‐region and the entire mature enzyme of the bee venom phospholipase A2 precursor. This sequence differs in the central region from the one determined by Shipolini et al. [FEBS Lett. 17, 39–40 (1971)] in showing, among other exchanges, two additional cysteines. The revised sequence of bee venom phospholipase is similar to the pancreatic enzyme in the spacing of cysteines and the presence of several amino acids known to be part of the active site or the Ca2+‐binding region in identical positions. Moreover, these parts of the bee protein can be fitted into the three‐dimensional structure determined for the bovine pancreatic phospholipase A2 [Dijkstra et al.(1981) Nature 289, 604–606]. Contrary to earlier suggestions, we therefore conclude that the bee venom enzyme shows some homology to phospholipases from mammalian pancreas and snake venoms.
Structural changes in positively and negatively supercoiled DNA
Authors: BRAHMS Sabine , NAKASU Syo , KIKUCHI Aki , BRAHMS J. Georges
Published: March 2005

The effect of superhelical constraint on the structure of covalently closed circular DNA (cccDNA; pBR322) with positive and negative writhe (superturn) has been investigated as a function of decreasing and increasing specific linking difference (mean superhelical density σ¯). At low and moderate negative superhelical densities σ¯, the overall average structure is maintained in an unwound B‐form slightly modified.

The overwound cccDNAs with positive writhe differ from those with negative writhe by an absence of cruciform structure. At high negative densities of supercoiling different changes involving the reversal of twist handedness are shown to lead to the formation of DNA segments in a conformation identical to the left‐handed component of form V DNA.
Serological analysis and characterization of calf thymus ribonuclease H IIb
Authors: VONWIRTH Heiner , FRANK Peter , BÜSEN Werner
Published: March 2005

Ribonuclease H IIb, which seems to play a physiological role during transcription, was purified from calf thymus tissue. A polyclonal antibody, raised against the most purified ribonuclease H IIb fraction, recognizes in crude extracts almost exclusively a 52‐kDa protein band. By immunoaffinity chromatography and immunoprecipitation experiments, we are able to deplete enzyme extracts from the crossreacting 52‐kDa protein band and from ribonuclease H IIb activity. Enzyme activity is eluted from the immunoaffinity matrix in association with a 52‐kDa protein under denaturing conditions. Immunoaffinity chromatography enables us also to calculate a purification factor of around 20000 from the crude extract. The native molecular mass for the enzyme of around 45 kDa, as determined by gel filtration, suggests that calf thymus ribonuclease H IIb is most probably monomeric.

The enzyme possesses an isoelectric point of 7.0. It requires Mg2+ ions for activity, is inhibited by N‐ethylmaleimide, and exhibits a pH optimum of 9.0–9.5. The enzyme releases oligoribonucleotides with 3′‐OH and 5′‐phosphate ends, probably in an exonucleolytical manner.

The third largest subunit of yeast RNA polymerase A (I) displays ribonuclease H activity [Huet et al. (1976) Nature 261, 431–433]. We discuss our findings in the light of a possible association of ribonuclease H IIb and RNA polymerase A (I) in higher eukaryotes.
Specific binding of bone sialoprotein to Staphylococcus aureus isolated from patients with osteomyelitis
Authors: RYDÉN Cecilia , YACOUB Alia Ibrahim , MAXE Ingemar , HEINEGÅRD Dick , OLDBERG Åke , FRANZÉN Ahnders , LJUNGH Åsa , RUBIN Kristofer
Published: March 2005

Bone sialoprotein is selectively bound by Staphylococcus aureus cells isolated from patients suffering from infections of bone tissue [Rydén, C., Maxe, I., Franzén, A., Ljungh, Å., Heinegård, D. & Rubin, K. (1987) Lancet II, 514]. In the present communication the binding of bone sialoprotein to staphylococcal cells is characterized in more detail. 125I‐Labelled bone sialoprotein bound to suspended staphylococcal cells in a time‐dependent, saturable and reversible manner. Binding was inhibited by unlabelled bone sialoprotein and by an amino‐terminal CNBr fragment of bone sialoprotein that did not contain the eukaryotic cell‐binding site. Binding was furthermore inhibited by lysates obtained from Escherichia coli lysogens carrying a λ gt11 phage‐encoding bone sialoprotein. In contrast, binding was not inhibited by a bacterial lysate from an osteopontin λ gt11 lysogen, nor by N‐linked oligosaccharide isolated from bone sialoprotein or by proteoglycan from rat chondrosarcoma containing clustered O‐linked oligosaccharides of the same structure as those of bone sialoprotein. These results indicate that the major staphylococcal‐binding site resides in the bone sialoprotein core protein and not in the carbohydrate side chains. No inhibition of bone sialoprotein binding could be detected for whole human serum or purified plasma proteins such as fibronectin, fibrinogen and IgG. Likewise, staphylococcal protein A or rat collagen type I did not inhibit the binding of bone sialoprotein. The latter results indicate that the binding site for bone sialoprotein on staphylococcal cells was not any of the hitherto described staphylococcal cell‐surface proteins. Binding data indicated an average of 1000 bone‐sialoprotein‐binding sites/bacterial cell.
Isolation and characterisation of a cDNA clone for a chlorophyll synthesis enzyme from Euglena gracilis
Authors: SHARIF Abid L. , SMITH Alison G. , ABELL Christopher
Published: March 2005

A cDNA expression library was constructed from light‐grown Euglena gracilis poly(A)‐rich RNA in λgt11. Antibodies to Euglena hydroxymethylbilane synthase, the third enzyme in the porphyrin biosynthetic pathway, were used to screen the library and a clone encoding part of the sequence of hydroxymethylbilane synthase was identified. This was used to rescreen the library and a full‐length clone was isolated, which encoded not only the entire mature protein (Mr 36 927), but also an N‐terminal extension of 139 amino acids. The deduced Mr of the whole polypeptide is 51 744, which corresponds to the size of the protein immunoprecipitated from the translation products of Euglena poly(A)‐rich RNA. The mature protein is 60–70% similar to hydroxymethylbilane synthase from human erythrocytes and Escherichia coli. The sequence of the N‐terminal extension has similarities to both the transit peptides of chloroplast proteins and those for the endoplasmic reticulum. This is the first report both of a cDNA clone for an enzyme of the chlorophyll biosynthetic pathway and of a putative transit peptide for a nuclear‐encoded Euglena protein.
Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells
Authors: SALLAFRANQUE‐ANDREOLA Marie‐Line , ROBERT Dominique , BARR Philip J. , FOURNIER Michel , LITVAK Simon , SARIH‐COTTIN Leila , TARRAGO‐LITVAK Laura
Published: March 2005

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast‐expressed reverse transcriptase [Barr, P. J., Power, M. D., Chun Ting Lee‐Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486–489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N‐ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.
A structural study of bovine lens α‐crystallin and its subunits by absorption and linear dichroism spectroscopy
Authors: BLOEMENDAL Michael , van AMERONGEN Herbert , BLOEMENDAL Hans , van GRONDELLE Rienk
Published: March 2005

The structure of the bovine α‐crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250–350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in α‐crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of α‐crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.
Phosphorylation of the high‐mobility‐group‐like protein P1 by casein kinase‐2
Authors: MÆLANDSMO Gunhild M. , ØSTVOLD Anne C. , LALAND Søren G.
Published: March 2005

The nuclear protein P1 (molecular mass 53 kDa), found in all mammalian cell types and tissues so far tested, is an excellent substrate for casein kinase‐2. The number of phosphate groups on P1 is 20–30/molecule; the phosphorylation sites are distributed throughout the molecule. The phosphate is present as serine phosphate and possibly threonine phosphate. Proteolytic digestion with Staphylococcus aureus V8 protease of 32P‐labelled P1 both in vivo and in vitro revealed that casein kinase‐2 may be one of the kinases responsible for the phosphorylation in vivo.
An NMR analysis of the binding of inhibitors to yeast phosphoglycerate kinase
Authors: BOYLE Helena A. , FAIRBROTHER Wayne J. , WILLIAMS Robert J. P.
Published: March 2005

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.
Purification and molecular properties of rabbit liver esterase ES‐1A
Authors: van LITH Hein A. , BIEMAN Maria den , van ZUTPHEN Bert F. M.
Published: March 2005

The isolation of esterase ES‐1A from rabbit liver microsomes/lysosomes is reported. The purification, as measured by methylbutyrate‐hydrolysing activity, was about 27‐fold with a recovery of 2.4%.

The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing.

The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size.

ES‐1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1).

The high degree of similarity of the properties of rabbit ES‐1A with those of mouse ES‐6A and rat ES‐10 suggests that these three esterases may have a common evolutionary origin.
Molecular and cellular studies of tryptophanyl‐tRNA synthetase using monoclonal antibodies
Authors: BERESTEN Sergey F. , ZARGAROVA Tatyana A. , FAVOROVA Ol'ga O. , RUBIKAITE Birute I. , RYAZANOV Alexey G. , KISSELEV Lev L.
Published: March 2005

Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl‐tRNA synthetase were prepared.

Am2 antibodies inhibit the Trp‐tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40‐kDa or 51‐kDa fragments) produced by limited proteolysis. Am1 and Am3 antibodies exert no effect on the Trp‐tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp‐tRNA synthetase molecule. Am1 cross‐reacts with Trp‐tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis.

A rapid two‐step technique was developed for isolating electrophoretically homogeneous Trp‐tRNA synthetase from Escherichia coli. The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations. As in the case of eukaroytic Trp‐tRNA synthetase, Am1 did not influence the activity of Trp‐tRNA synthetase from E. coli.

From the aforementioned results it follows that: (a) the conservation of part of the Trp‐tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp‐tRNA synthetases suggests that the dispensalbe part of the molecule might be involved in some additional biological function(s) of Trp‐tRNA synthetase besides tRNATrp charging; (b) the common antigenic determinant in Trp‐tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.
Molecular and cellular studies of tryptophanyl‐tRNA synthetases using monoclonal antibodies
Authors: FAVOROVA Olga O. , ZARGAROVA Tatyana A. , RUKOSUYEV Vadim S. , BERESTEN Sergey F. , KISSELEV Lev L.
Published: March 2005

The content of Trp‐tRNA synthetase in pancreas and liver of cattle, sheep, swine, rat, rabbit and man was assayed by direct radioimmunoblotting with a 125I‐labelled monoclonal antibody Am1, specifically interacting with any cukaryotic Trp‐tRNA synthetase. Its content in the organs studied, with the exception of bovine and sheep pancreas, was found to be 0.002–0.012% of total proteins. The enzyme content in bovine pancreas was about 0.2% of total proteins, i.e. 70 times higher than in bovine liver; similar correlations were found for sheep. The Trp‐tRNA synthetase levels in each organ varied from animal to animal of the same species by not more than a factor of four; these individual variations cannot affect the conclusion about the profound differences in the levels of the enzyme in pancreases of Ruminantia and of the other mammalians.

As shown by indirect immunofluorescence technique, bovine Trp‐tRNA synthetase is mainly located in the exocrine part of the pancreas. Moreover, the immunoreactive material is detectable also in bovine (not human) pancreatic juice. The abnormally high Trp‐tRNA synthetase content in the ruminant pancreas may be connected with unknown function(s) of this protein somehow related to the peculiarities of digestion of these mammals.