bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of a Coenzyme Analog with Aspartate Aminotransferase Isoenzymes in the Crystal
Authors: OTTONELLO Simone , MOZZARELLI Andrea , ROSSI Gian Luigi , CAROTTI Daniela , RIVA Francesca
Published: March 2005

The interaction between the coenzyme derivative 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate with cytoplasmic and mitochondrial apo‐aspartate aminotransferase in the crystalline state was investigated to establish whether the structural differences, known to exist between the active sites of the two isoenzymes in solution, are maintained in the crystal although they are not apparent from the available crystallographic data.

In the crystal, as in solution, both apo‐isoenzymes reversibly bind the coenzyme derivative and catalyze a slow cleavage reaction, by which pyridoxal 5′‐phosphate is produced and bound to the active‐site lysine. In the case of the cytoplasmic isoenzyme, however, in the crystal as in solution, the initial complex can follow an alternative reaction path that leads to the formation of a covalent bond between the active‐site lysine and the C‐5 of the 2,4‐dinitrophenyl moiety of the reagent.

Therefore, crystal‐packing forces neither abolish the active site properties that are needed to cleave the specifically bound reagent and are common to the two isoenzymes nor mask the subtle differences that allow for the selective irreversible labeling of the cytoplasmic isoenzyme.
The Refined Structure of the Selenoenzyme Glutathione Peroxidase at 0.2‐nm Resolution
Authors: EPP Otto , LADENSTEIN Rudolf , WENDEL Albrecht
Published: March 2005

The crystal structure of bovine erythrocyte glutathione peroxidase has been refined by a combined procedure of restrained crystallographic refinement and energy minimization at 0.20 nm resolution. The final R value at this resolution is 0.178. The r.m.s. deviation of main‐chain atoms of the two independently refined monomers is 0.019 nm. The structure at 0.28 nm resolution, which has been determined by multiple isomorphous replacement, served as a starting model.

The refined model allowed a detailed survey of the hydrogen‐bonding pattern and of the subunit contact areas in the molecule. The model contains 165 solvent molecules per dimer, all taken as water molecules. The mobility of the structure was derived from the individual atomic temperature factors. The complete tetramer, including the active sites, seems to be rather rigid, except for narrow loops near to the N‐terminal ends and some β turns exposed to solvent.

The active centres of glutathione peroxidase are found in flat depressions on the molecular surface. The catalytically active selenocysteine residues could be located at the N‐terminal ends of α helices forming βαβ substructures together with two adjacent parallel β strands. In the vicinity of the reactive group some aromatic amino acid side‐chains could be localized. Especially Trp‐148, which could be hydrogen bonded to SeCys‐35, may play a functional role during catalysis.

The results of substrate and inhibitor binding studies in solution and in the crystalline state could be interpreted by an apparent half‐site reactivity of glutathione peroxidase. The enzyme seems to react in the sense of negative cooperativity with dimers being the functional units.

Based on difference Fourier analyses of appropriate derivatives a reasonable model of glutathione binding is presented. Among the residues which could be of functional importance are Arg‐40, Gln‐130 and Arg‐167, presumably forming salt bridges and a hydrogen bond to the glutathione molecule.

In conclusion, a general picture of a minimal reaction mechanism, which is in good agreement with functional and structural data, is proposed. The main reaction of the catalytic cycle presumably shuttles between the selenolate and the selenenic acid state of SeCys‐35.
p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens
Authors: WEIJER Wicher J. , HOFSTEENGE Jan , BEINTEMA Jaap J. , WIERENGA Rik K. , DRENTH Jan
Published: March 2005

The complete primary and tertiary structure of p‐hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron‐density map at 0.25‐nm resolution. The parts of the polypeptide chain contributing the residues to the FAD‐binding site and the residues of the substrate‐binding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p‐hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD.

Many side chains from the C‐terminal part of the polypeptide chain are involved in subunit‐subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.
Purification and Properties of 2‐Aminoethylphosphonate: Pyruvate Aminotransferase from Pseudomonas aeruginusa
Authors: DUMORA Catherine , LACOSTE Anne‐Marie , CASSAIGNE André
Published: March 2005

2‐Aminoethylphosphonate aminotransferase has been purified to homogeneity with a yield of 15% from cell extracts of Pseudomonas aeruginosa. The molecular weight of the enzyme was estimated by gel filtration to be 65000 ± 2000. Sodium dedecyl sulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 16500 ± 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280 nm, 335 nm and 415 nm which are characteristic of a pyridoxal‐phosphate‐dependent enzyme: 4 mol of pyridoxal 5′‐phosphate/mol of enzyme have been found.

This aminotransferase catalyzes the transfer of the amino group of 2‐aminoethylphosphonate (ciliatine) to pyruvate to give 2‐phosphonoacetaldehyde and alanine. A pH optimum between 8.5–9 and an activity increasing from 30°C to 50°C have been observe. The reaction follows Michaelis‐Menten kinetics with Km values of 3.85 mM and 3.5 mM for ciliatine and pyruvate respectively.

This enzyme shows a very high specificity since ciliatine and pyruvate are pyruvate are the only amino donor and acceptor respectively. Methyl, ethyl and propylphosphonic acids are better competitors towards ciliatine than their α‐amino derivatives. 3‐Aminopropylphosphonate, the higher homologue of ciliatine, is recognized neither as a substrate nor as an inhibitor. The enzyme activity is significantly affected by carbonyl reagents and by HgCl2.

Transamination of 2‐aminoethylphosphonate is the first step of a double‐step pathway which leads to the cleavage of its C‐P bond.
Study of the Positive Control of the General Amino‐Acid Permease and Other Ammonia‐Sensitive Uptake Systems by the Product of the NPR1 Gene in the Yeast Saccharomyces cerevisiae
Authors: GRENSON Marcelle
Published: March 2005

Mutations at the NPR1 genetic locus are known to inactivate (totally or partially) at least five distinct ammonia‐sensitive permeases. Mutants with thermosensitive NPR1 gene product (nprts) have been used to discriminate between three possible roles of this protein, namely (a) a common constituent of a set of ammoni‐sensitive permeases; (b) a common activator of these permeases; (c) a common positive factor necessary for their synthesis.

Inactivation of the general amino‐acid permease was observed upon transfer of nprts mutant cells to a non‐permissive temperature. Under the same conditions, the general amino‐acid permease of the wild‐type cells remained active for several hours even when protein synthesis was inhibited by nitrogen starvation or by cycloheximide.

Mutations at three unlinked loci, namely the PGR site (located in the GAP1 structural gene of the permease), and the unlinked MUT2 and MUT4 loci restore the general amino‐acid permease activity in npr1 mutants.

The results are interpreted as indicating that the NPR1 product is necessary for the reactivation of the general amino‐acid permease which seems to be continuously inactivated by a regulatory process mediated by the MUT2 and the MUT4 gene products acting at the level of the PGR site of the general amino‐acid permease molecule.

The proline permease and the ureidosuccinic‐acid permease seem to be subject to the same double regulation by inactivation‐reactivation of the permeases and by repression of their synthesis.

A tentative scheme of the regulation of the general amino‐acid permease is presented.
The αβ‐Methylene Analogues of ADP and ATP Act as Substrates for Creatine Kinase
Authors: MILNER‐WHITE E. James , RYCROFT David S.
Published: March 2005

The αβ‐methylene analogues of ATP and ADP, [αβCH2]ATP and [αβCH2]ADP, are substrates for creatine kinase. However, the rate of the phosphoryl transfer reaction catalysed is about 10‐5‐times lower than that with normal ATP. The affinities of the analogues (especially [αβCH2ADP) for the enzyme are lower than those of the normal substrates. The equilibrium constant at 25°C, measured using 31P NMR, for the reaction Mg[αβCH2]ATP + creatine ⇌ Mg[αβCH2]ADP + phosphocreatine + H+ is 2.2 × 10‐12 M compared with a value of 2.5 × 10 ‐10 M for the same reaction with the normal substrates, corresponding to a difference in ΔG0 values of 11.7 kJ · mol−1. It follows that ΔG0 for the hydrolysis of the terminal phosphate group of Mg[αβCH2]ATP is less favourable by 11.7 kJ · mol−1 than that for MgATP.
The Relationship between Extramitochondrial Ca2+ Concentration, Respiratory Rate, and Membrane Potential in Mitochondria from Brown Adipose Tissue of the Rate
Authors: NEDERGAARD Jan
Published: March 2005

The ability of isolated mitochondria from rat brown‐adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, Δψ (estimated with safranine), and was varied semi‐physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32‐kDa polypeptide, thermogenin, which regulates coupling.

Ca2+ uptake (at 5 μM extramitochondrial Ca2+) was maximal at Δψ > 150mV. Basal Ca2+ release increased from 1 to 2 nmol × min−1× mg −1 below 150 mV. Na+‐stimulated rate of Ca2+ release was stable within the investigated Δψ span (100–160 mV).

Initial Ca2+ levels were maintained below 0.2 μM for 100mV < Δψ < 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+× mg−1 were below 0.4μM for Δψ > 135 mM.

Respiration was unstimulated for Δψ > 150 mV and was maximal at Δψ≤ 135mV. In the presence of well‐oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity–i.e. by a limited H+ reentry (OH− exit) and therefore by a membrane potential maintained at about 135 mV.

It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does decrease below 135 mV in vivo.

Therefore, Ca2+ homeostasis and thermogenesis are non‐interfering and can be hormonally independently regulated, e.g. by α‐adrenergic and β‐adrenergic stimuli, respectively.
Localisation of the Active Site of Pyruvate Carboxylase by Electron Microscopic Examination of Avidin‐Enzyme Complexes
Authors: JOHANNSSEN Walther , ATTWOOD Paul V. , WALLACE John C. , KEECH D. Bruce
Published: March 2005

Negatively stained enzyme‐avidin complexes, seen at different stages of the titration of the biotin‐binding sites on avidin with chicken liver pyruvate carboxylase, have been studied using electron microscopy. Formation of linear, unbranched polymers of the enzyme‐avidin complex is seen to occur when the ratio of avidin to enzyme is between 2:1 and 1:2; beyond these limits only single enzyme tetramers are visible. The single avidin molecules observed seem to have a cuboid structure. The orientation and dimensions of the enzyme tetramers within the polymers indicate that the tetrahedron‐like structure, observed in the single molecules, has been prserved. From the structure of the polymers and the observation of single enzyme‐avidin complexes, we deduce that the biotin groups on the enzyme are located on the external faces of each subunit close to, and probably within 3 nm of, the intersubunit junction.
Isolation and Characterization of Multiforms of Aldehyde Reductase in Chicken Kidney
Authors: HARA Akira , DEYASHIKI Yoshihiro , NAKAYAMA Toshihiro , SAWADA Hideo
Published: March 2005

Three multiforms of NADPH‐dependent aldehyde reductase have been purified to homogeneity from chicken kidney. The enzymes were monomeric proteins with similar molecular weights around 39000 but with different pI values. Two of them exhibited, almost the same heat stability, a broad optimal pH of 6–6.5 and preference for NADPH as a cofactor. They were high‐Km aldehyde reductases which reduced various aldehydes, hexonates, α‐diketones, xylose, glucuronolactone and ethyl acetoacetate, and which were inhibited to similar degrees by heavy metal ions, organic acids, p‐chloromercuribenzoate, indomethacin and phenobarbital. The third aldehyde reductase was distinct from the other two enzymes in its heat lability, sharp acidic pH optimum of 6.0, and dual cofactor specificity. The enzyme showed low Km values for the above‐mentioned substrates except hexonates and 4‐carboxybenzaldehyde, and it specifically reduced lauryl aldehyde, acetoin and ketosteroids. This enzyme was less sensitive to the above‐mentioned inhibitors than the high‐Km aldehyde reductases, but the enzyme was moderately inhibited by chlorpromazine and was activated about 1.5 times by the addition of sulfate ions.

It was demonstrated using antibodies against one of the high‐Km forms and the low‐Km form of aldehyde reductase that the two high‐Km forms were immunologically identical with each other but not with the low‐Km form, and that the high‐Km and low‐Km forms were widely distributed in various tissues of chicken.
Synthesis of α1‐Microglobulin by Guinea‐Pig Liver
Authors: ÅKERSTRÖM Bo
Published: March 2005

Explants from perfused guinea‐pig livers were found to release α1‐microglobulin into the culture medium. Tritiated leucine in the medium was incorporated into the protein, suggesting a de novo synthesis of α1‐microglobulin by the liver tissue. The size of the protein could not be distinguished from that of purified urinary α1‐microglobulin when tested with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After intravenous injections of tritiated leucine into guinea‐pigs, the 105000 ×g pellet of homogenized liver rapidly increased its content of radioactive α1‐microglobulin, with a maximum after 20 min. 3H‐Labelled α1‐microglobulin appeared in serum after a lag phase of 20 min, and by comparing the rate of accumulation with albumin, the synthesis of guinea‐pig α1‐microglobulin could be estimated to 20 μg g liver−1 h−1.
Hepatocyte Heterogeneity in Glutamine and Ammonia Metabolism and the Role of an Intercellular Glutamine Cycle during Ureogenesis in Perfused Rat Liver
Authors: HÄUSSINGER Dieter
Published: March 2005

The metabolism of glutamine and ammonia was studied in isolated perfused rat liver in relation to its dependence on the direction of perfusion by comparing the physiological antegrade (portal to caval vein) to the retrograde direction (caval to portal vein).

Added ammonium ions are mainly converted to urea in antegrade and to glutamine in retrograde perfusions. In the absence of added ammonia, endogenously arising ammonium ions are converted to glutamine in antegrade, but are washed out in retrograde perfusions. When glutamine synthetase is inhibited by methionine sulfoximine, direction of perfusion has no effect on urea synthesis from added or endogenous ammonia.

14CO2 production from [1‐14C]glutamine is higher in antegrade than in retrograde perfusions as a consequence of label dilution during retrograde perfusions.

The results are explained by substrate and enzyme activity gradients along the liver lobule under conditions of limiting ammonia supply for glutamine and urea synthesis, and they are consistent with a perivenous localization of glutamine synthetase and a predominantly periportal localization of glutaminase and urea synthesis. Further, the data indicate a predominantly periportal localization of endogenous ammonia production. The results provide a basis for an intercellular (as opposed to intracellular) glutamine cycling and its role under different metabolic conditions.
Characterization of Messenger RNA for Chick‐Embryo DNA Polymerase β and Its Translation Product in vitro
Authors: YAMAGUCHI Masamitsu , TAKAHASHI Taijo , YASUDA Kunio , SHIMURA Yoshiro , MATSUKAGE Akio
Published: March 2005

A specific immunoprecipitation method, using rabbit anti‐(chick DNA polymerase β) IgG was applied to detect the polypeptide of DNA polymerase β among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr= 40 000 was specifically immunoprecipitated from [35S]methionine‐labeled translation products and was competitive with the purified DNA polymerase β for the antibody. Furthermore, the 40 000‐Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase β was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)‐rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.
Acyl‐Coenzyme A: Cholesterol Acyltransferase
Authors: SYNOURI‐VRETTAKOU Stella , MITROPOULOS Konstantinos A.
Published: March 2005

The preincubation at 37°C of rat liver microsomal fraction, followed by re‐isolation of the treated vesicles, results in a time‐dependent increase in the activity of acyl‐CoA: cholesterol acyltransferase. The presence of cholesterol‐phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration‐dependent.

The preincubation of the microsomal fraction in the presence of [3H]cholesterol‐phospholipid liposomes results in transfer of [3H]cholesterol to the re‐isolated microsomal vesicles and this transfer follows first‐order kinetics in respect to the donor concentration. These preincubations result also in a time‐dependent and liposomal cholesterol concentration‐dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half‐time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltranferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time‐dependent and temperature‐dependent increase in cholesterol acyltransferase activity is due to the transfer of non‐esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.
The Phytochrome‐Controlled Accumulation of mRNA Sequences Encoding the Light‐Harvesting Chlorophyll a/b Protein of Barley (Hordeum vulgare L.)
Authors: GOLLMER Ingrid , APEL Klaus
Published: March 2005

Double‐stranded cDNA was synthesized from polysomal poly(A)‐containing RNA of illuminated barley plants and was inserted into the PstI site of the bacterial plasmid pBR322. Two different strategies were used for the screening of the bacterial colonies. Light‐regulated sequences were detected by differential hybridization with cDNA of polyadenylated RNA from dark‐grown and illuminated barley plants. For a second screening step partially purified mRNAs encoding the light‐harvesting chlorophyll a/b protein were used for the synthesis of another cDNA probe. By using these procedures a cDNA clone was isolated which encodes a constitutive polypeptide of the light‐harvesting chlorophyll a/b protein.

This cloned cDNA has been used to assess the effect of phytochrome on the steady‐state level of mRNA sequences encoding the light‐harvesting chlorophyll a/b protein. The mRNA is almost undetectable in dark‐grown plants. Following treatment with red light, the concentration of this mRNA sequence increases rapidly during the subsequent dark period. This red light effect can be reversed substantially by irradiation with far‐red light. These results indicate that not only the amount of mRNA activity as shown previously, but also the steady‐state level of mRNA sequences encoding chrome. the light‐harvesting chlbrophyll a/b is contrdlled by phytochrome.
Neutron Diffraction of Chromatin in Interphase Nuclei and Metaphase Chromosomes
Authors: IBEL Konrad , KLINGHOLZ Reiner , STRÄTLING Wolf H. , BOGENBERGER Jakob , FITTLER Friedrich
Published: March 2005

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration.

Aliquots of a suspension of rat liver nuclei prepared in a polyamine‐free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KC1; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (l/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer‐filled spaces with an overall size of the order of 35 ‐ 45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosonial particles with an overall diameter of 40–50 nm. The compact chromatin structure disassembled directly into these fundamental structural units.

Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.
Secretion of Plant Storage Globulin Polypeptides by Xenopus laevis Oocytes
Authors: BASSUÜNER Ronald , HUTH Antje , MANTEUFFEL Renate , RAPOPORT Tom A.
Published: March 2005

Xenopus oocytes injected with poly(A)‐containing RNA from developing cotyledons of field beans (Vicia fizha L. var. minor) synthesize precursor polypeptides to the major storage globulins legumin and vicilin. These polypeptides are secreted into the medium without proteolytic cleavage of the legumin propolypeptides into the mature disulfide‐linked α and β chains. Similarly, storage globulin polypeptides from pea (Pisum sativum L.) and french bean (Phaseolus vulgaris L.) were secreted from oocytes. Inhibition of glycosylation by tunicamycin does not prevent secretion. This first report on the secretion of plant polypeptides by Xenopus oocytes shows that (a) intracellular deposition of storage proteins in membrane‐bounded organelles (protein bodies) of plants and extracellular secretion have step(s) in common, and (b) the cell, in addition to the mRNA, determines the final destination of these polypeptides.
On the Probable Involvement of Arginine Residues in the Bile‐Salt‐Binding Site of Human Pancreatic Carboxylic Ester Hydrolase
Authors: LOMBARDO Dominique , CAMPESE Daniel , MULTIGNER Luc , LAFONT Huguette , DE CARO Alain
Published: March 2005

Modification of arginine residues with 2,3‐butanedione inhibits the carboxylic‐ester hydrolase activity on soluble and emulsified substrates when assayed with bile salts. The α‐dicarbonyl reagent modifies seven of the nineteen arginine residues present per enzyme molecule. Nevertheless the inactivation with butanedione is greatly diminished when the protein is in the presence of negatively charged micellar bile salt. In these conditions we observe the protection of one arginine residue by sodium taurodeoxycholate and of two arginine residues by sodium cholate. This suggests that the carboxylic‐ester hydrolase from human pancreatic juice contains at least two arginine residues essential for the activation by bile salts. All our data confirm the presence of two bile‐salt‐binding sites on the enzyme in which one arginine per site is involved and plays the general role of an anionic binding site. This study provides evidence that arginine residues may play an essential role in the interaction between bile salts and protein.
Transport of Methionine in Sea‐Urchin Sperm by a Neutral Amino‐Acid Carrier
Authors: GACHE Christian , VACQUIER Victor D.
Published: March 2005

A carrier‐mediated transport for l‐methionine and other neutral amino acids exists in sperm of the sea urchin Lytechinus pictus. The initial rate of l‐methionine entry is a Michaelis‐Menten function of the methionine concentration in the external medium. The maximum velocity is low [V = 250 pmol h−1 (109 sperm)−1 at 22 °C] and the affinity is high (Km= 6–10 μM). The initial rate of transport under steady‐state exchange conditions is also a Michaelis‐Menten function of the external concentration of methionine. The Km determined by this method is about 14 pM. Neutral amino acids compete with l‐methionine transport as shown by initial velocity measurements. These results indicate that l‐methionine transport is a carrier‐mediated process. The temperature dependence of the process is approximately 84 kJ (20 kcal) mol−1 K−1, which is not compatible with a simple diffusion mechanism, but in the range of values usually found for a mediated transport. The transport is largely Na+‐independent and does not depend on Ca2+, K+ or HC gradients. It is only partially sensitive to KCN, showing it is mainly independent of oxidative phosphorylation. The steady‐state internal methionine concentration is not a linear function of the external amino acid concentration. This suggests that an exit by diffusion competes with a carrier‐mediated concentrative transport in a cellular compartment. This mediated transport is compared to those of higher animal cells.
Partial Purification of Rat and Human Serum Factors Inhibiting the G1‐S Transition in Rat Hepatocytes
Authors: AUGER Geneviève , BLANOT Didier , VAN HEIJENOORT Jean , NADAL Claude , GOURNAY Marie‐Françoise
Published: March 2005

A low molecular weight compound, which inhibits the G1‐S transition in rat hepatocytes, was purified from rat trypsin‐treated serum by DEAE‐cellulose chromatography and high‐performance liquid chromatography on three different stationary phases. This procedure led to a 34500‐fold purification with a 29% yield. Inactivation of the purified material by specific enzymes showed that the inhibitor is a glycopeptide containing a peptide moiety, N‐ acetylneuraminic acid and galactose residues. Amino acid analyses indicated the possible existence of a pentapeptide structure. The same purification procedure was applied to the corresponding human inhibitor. Inactivation by specific enzymes showed that it is also a glycopeptide.
Neutron‐Scattering Studies of Accurately Reconstituted Nucleosome Core particles and the Effect of Ionic Strength on Core Particle Structure
Authors: SIBBET Gray J. , CARPENTER Brian G. , IBEL Konrad , MAY Roland P. , KNEALE G. Geoffrey , BRADBURY E. Morton , BALDWIN John P.
Published: March 2005

Chicken erythrocyte nucleosome core particles can be dissociated quantitatively into histone (H3, H4)2 bound to 146 base pairs of DNA, and 2(H2A, H2B). Reconstitution of core particles from the two components produces an 85% yield of particles which neutron scattering studies show to be accurate stoichiometrically and indistinguishable from native core particles: the radii of gyration of the shape, the protein components and the DNA components of the particles are 4.02 nm, 3.3 nm and 4.95 nm respectively. The largest distance and most probable distance which can be drawn in the particles are 11.5 nm and 4.3 nm respectively. The molecular weight of the particles is identical to that of control ‘native’ core particles. All of these values, within limits of error, are the same as known values for ‘native’ core particles. These experiments confirm the essential role of histones H3 and H4 in the initial organisation of core‐particle structure, make possible the manufacture of perfectly pure and homogeneous core‐particle preparations and allow the 100% incorporation of labelled or modified histones.

Neutron scattering studies of core particles at high contrats (in D2O and H2O) have been carried out over a range of ionic strengths and pH. No change in structure is detected down to pH 5.5 in 20 mM NaCl or down to ionic strength 2.0 mM at pH 7.
Expression of Interferon‐α and Interferon‐β Genes in Human Lymphoblastoid (Namalwa) Cells
Authors: SHUTTLEWORTH John , MORSER John , BURKE Derek C.
Published: March 2005

Treatment of human lymphoblastoid (Namalwa) cells with Sendai virus induced the coordinate synthesis of both IFN‐α and IFN‐β interferon mRNAs. One sub‐line of Namalwa cells (WRL) produced no IFN‐β activity, although IFN‐β mRNA was induced and was associated with polysomes. The IFN‐α mRNA was heterogeneous, ranging in size over 1.20–1.35 × 103bases, probably because of variation in the size of the transcribale DNA in the α‐gene family. The IFN‐β mRNA was monodisperse with a size of 1.05 × 103bases. The kinetics of accumulation and decay of both IFN‐α and IFN‐β mRNAs, as assessed by hybridization with cDNA probes, were very similar to those of translatable interferon mRNA, as assessed by translation inXenopusoocytes.

Treatment of the cells with butyrate or 5′‐bromodeoxyuridine increased the amount of hybridizable IFN‐α and IFN‐β mRNA about 15‐fold and 4‐fold respectively, again demonstrating coordinate control of IFN‐α and IFN‐β production.
Characterisation of a Reconstituted Mg‐ATP‐Dependent Protein Phosphatase
Authors: RESINK Therese J. , HEMMINGS Brian A. , TUNG H. Y. Lim , COHEN Philip
Published: March 2005

Homogenous preparations of the catalytic subunit of protein phosphatase‐1 and inhibitor‐2 can be combined to produce an inactive enzyme that consists of a 1:1 complex between these two proteins. This species is indistinguishable from the Mg‐ATP‐dependent protein phosphatase in that preincubation with glycogen synthase kinase‐3 and Mg‐ATP is required to generate activity. Activation results from the phosphorylation of inhibitor‐2. The molar concentrations of protein phosphatase‐1 and inhibitor‐2 in rabbit skeletal muscle (0.25–0.5 μM) are similar.

Incubation of the reconstituted Mg‐ATP‐dependent protein phosphatase with chymotrypsin is accompanied by limited proteolysis of inhibitor‐2 and the loss of its phosphorylation site(s). This species can be activated by glycogen synthase kinase‐3 and Mg‐ATP provided that inhibitor‐2 is added. This exogenous inhibitor‐2 appears to displace the fragments of inhibitor‐2 from the enzyme that were generated by chymotryptic digestion. These experiments may explain the report [Yang, S. D., Vandenheede, J. R. and Merlevede, W. (1981) J. Biol. Chem. 256, 10231–10234] that inhibitor‐2 can function as an ‘activator’ as well as an inhibitor of the Mg‐ATP‐dependent protein phosphatase.

Incubation of the catalytic subunit of protein phosphatase‐1 with sodium fluoride or sodium pyrophosphate converted the enzyme to an inactive from that could be partially reactivated by manganese ions, but not by glycogen synthase kinase‐3 and Mg‐ATP. Conversely, the reconstituted Mg‐ATP‐dependent protein phosphatase could only be activated by glycogen synthase kinase‐3 and Mg‐ATP, and not by manganese ions. It is concluded that the conversion of protein phosphatase‐1 to a manganese‐ion dependent form is a quite separate phenomenon from the formation of the Mg‐ATP‐dependent protein phosphatase.

Inhibitor‐2 can inactivate protein phosphatase‐1 by a second mechanism that is not reversed by preincubation with glycogen synthase kinase‐3 and Mg‐ATP. This occurs at higher concentrations of inhibitor‐2 than those required to form the Mg‐ATP‐dependent protein phosphatase, and appears to result from the binding of inhibitor‐2 to a distinct site on the enzyme.
On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by 13C‐Nuclear‐Magnetic‐Resonance Relaxation
Authors: MOONEN Chrit T. W. , MÜLLER Franz
Published: March 2005

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non‐protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (>90%13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole‐dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein‐bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field‐dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP.

In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.
A Form of the DNA‐Dependent RNA Polymerase of Halobacterium halobium, Containing an Additional Component, Is Able to Transcribe Native DNA
Authors: MADON Jerzy , ZILLIG Wolfram
Published: March 2005

A revised procedure for the purification of DNA‐dependent RNA polymerase from Halobacterium halobium, including two‐phase partition, yields pure, highly active and absolutely DNA‐dependent enzyme. Two forms of the enzyme, one containing, the other not containing a previously not observed component, ɛ, show striking differences in activity. RNA polymerase without component ɛ has a significant activity on poly[d(A–T)] but only insignificant activity on all other templates. The enzyme containing a stoichiometric amount of component ɛ transcribes poly[d(A–T)] and native templates efficiently.
Sequence Requirement for Transcription in vitro of the Human Corticotropin/β‐Lipotropin Precursor Gene
Authors: NOTAKE Mitsue , KUROSAKI Tomohiro , YAMAMOTO Tokuo , HANDA Hiroshi , MISHINA Masayoshi , NUMA Shosaku
Published: March 2005

Using HeLa whole cell extracts, we have demonstrated that transciption in vitro of the cloned human and bovine corticotropin/β‐lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5′‐deletion mutants of a fusion gene that contains the 5′‐flanking sequence and capping site of the human corticotropin/β‐lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the ‘TATA box’ present in this region seems to be the main promoter element for transcription of the human corticotropin/β‐lipotropin precursor gene in the HeLa cell‐free system.

We have also developed a transcription system in vitro from the corticotropin‐producing mouse pituitary tumor cell line AtT‐20 in culture. Deletion mapping of the fusion gene promoter has indicated that the ‘TATA box’ region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT‐20 cell‐free system, but hardly in the HeLa cell‐free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT‐20 cell extract is responsible for the modulation of transcription of the human corticotropin/β‐lipotropin precursor gene.
Phytochrome‐Mediated Regulation of Two mRNAs, Encoded by Nuclei and Chloroplasts of Ribulose‐1,5‐bisphosphate Carboxylase/Oxygenase
Authors: SASAKI Yukiko , SAKIHAMA Toshiko , KAMIKUBO Tadashi , SHINOZAKI Kazuo
Published: March 2005

Etiolated pea seedlings (5 days old) were treated with red light for 5 min and grown for 2 days more in darkness. Effects of the red light on the mRNA levels of two subunits of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (ribulose‐P2 carboxylase), rate of enzyme synthesis and accumulation of the enzyme were examined for the plants. Increases of the two mRNA levels, encoded in nuclei and chloroplasts were observed. Red light followed by far‐red light treatment for 5 min inhibited the increases. An increase parallel with the mRNA levels was found for the rate of the enzyme synthesis and accumulation of the enzyme. These results indicate that the induction of two mRNA levels is mediated by phytochrome and causes the red‐light‐inducible, far‐red‐light‐reversible increase of ribulose‐P2 carboxylase. When white light was irradiated to induce the enzyme, the effects of red light given in darkness were reinforced. These results indicate that light‐induction of ribulose‐P2 carboxylase is mediated by phytochrome and controlled by the two mRNA levels.
Molecular Forms of β‐N‐Acetylhexosaminidase in Epstein‐Barr Virus‐Transformed Lymphoid Cell Lines from Normal Subjects and Patients with Tay‐Sachs Disease
Authors: SALVAYRE Robert , MARET Arlette , NEGRE Anne , LENOIR Gilbert , VUILLAUME Michèle , ICART Josette , DIDIER Jacqueline , DOUSTE‐BLAZY Louis
Published: March 2005

In whole leukocytes and in lymphocytes from normal subjects, the percentage activity of heat‐stable β‐N‐acetylhexosaminidase (30 ± 5% and 45 ± 5%, respectively) was higher than in the transformed lymphoid cell line (19 ± 3%). In Tay‐Sachs transformed cells as well as non‐transformed lymphocytes, β‐N‐acetylhexosaminidase was almost completely heat‐stable (95–98%).

In the transformed cells from normal subjects, the β‐N‐acetylhexosaminidase B (Hex B) activity (5% of total) was significantly lower than in blood lymphocytes (average 25–30% of total activity), whereas Hex A and Hex I were similar in the either cell type.

Blood lymphocytes and lymphoid cell lines established from a Tay‐Sachs patient lacked heat‐labile Hex A and expressed high heat‐stable Hex I and Hex B activities (3–6‐fold).

After neuraminidase treatment, Hex A peak sharpened while Hex I peaks switched to higher pI than normal Hex I, in the region of Hex B. PreHex A/S pI was not affected.

Hydrolytic properties using the both substrates (4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐glucopyranoside and 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐β‐d‐galactopyranoside) of each molecular form were similar in transformed and non‐transformed cells. Data derived from the use of a mixture of substrates were consistent with the model which proposes a common active site for either substrate in the case of preHex A, Hex B and Hex I, but not for Hex A.

Thus Epstein‐Barr virus‐transformed lymphoid cell lines represent an accurate model system for studies on Tay‐Sachs disease.
Synergism between Coenzyme and Aldehyde Binding to Liver Alcohol Dehydrogenase
Authors: ANDERSSON Pia , KVASSMAN Jan , OLDEN Bertil , PETTERSSON Gösta
Published: March 2005

Aldehyde binding to liver alcohol dehydrogenase in the absence and presence of coenzymes has been characterized by spectrometric equilibrium methods, using auramine O and bipyridine as reporter ligands.

Free enzyme shows a significant affinity for aldehydes, and equilibrium constants for dissociation of the binary complexes formed with typical aldehyde substrates are reported. Binary‐complex formation does not lead to any detectable inner‐sphere coordination of aldehydes to the catalytic zinc ion of the enzyme subunit.

Complex formation with NAD+ or NADH increases the affinity of the enzyme for aromatic aldehydes by a factor of 1.8–3.5 and 6–17, respectively. Benzaldehyde and dimethylaminocinnamaldehyde binding to the enzyme · NAD+ complex is not detectably associated with inner‐sphere coordination of the aldehyde to zinc.

It is concluded that binding of NADH is required to induce catalytically adequate bonding interactions between enzyme and aromatic aldehydes. The effect of reduced coenzyme in this respect is attributed to hydrophobic interactions leading to dehydration of the active‐site region, which allows aldehyde substrates to compete successfully with water for inner‐sphere coordination to the catalytic zinc ion. Oxidized coenzyme is proposed to have a similar promoting effect on metal coordination of aldehydes which function as substrates for the dismutase activity of the enzyme.
Aberrant Migration of Lipopolysaccharide in Sodium Dodecyl Sulfate/Poyacrylamide Gel Electrophoresis
Authors: HITCHCOCK Penny J.
Published: March 2005

Purified lipopolysaccharides of salmoellae strains were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Pre‐electrophorees of polyacrylamide gels had no apparent effect on one‐dimensional silver‐stained lipopolysaccharide profiles. However, without pre‐electrophoresis, two‐dimensional and three‐dimensional patterns contained numerous bands with varied migration patterns compared to those in the one‐dimension gels. The lipopolysaccharide was altered within the polyacrylamide gel during electrophoresis. Pre‐electrophoresis of gels eliminated aberrant migration patterns.
Induction of Differentiation in a Rat Mammary Epithelial Stem Cell Line by Dimethyl Sulphoxide and Mammotrophic Hormones
Authors: WARBURTON Michael J. , HEAD Linda P. , FERNS Sharon A. , RUDLAND Philip S.
Published: March 2005

Rama 25 is a clonal epithelial cell line derived from a dimethylbenzanthracene‐induced rat mammary adenocarcinoma. In the presence of the mammotrophic hormones, insulin, hydrocortisone, estrogen and prolactin, Rama 25 produces small amounts of casein and forms domes at a low rate. The rates of both these processes can be greatly increased by the addition of dimethyl sulphoxide or hexamethylenebisacetamide which are also known to induce the differentiation of Friend erythroleukemia cells. Other compounds which stimulate the differentiation of Rama 25 cells include linoleic acid and 6‐thioguanine. The intracellular pathways triggering changes in the two markers of differentiation are partially separable using different combinations of hormones, prolactin and hydrocortisone being the most important for the production of casein and the formation of domes respectively. The kinetics of differentiation, as judged by the appearance of these two markers, are characterised by two phases, a fixed period of 8 h (lag phase), the length of which is independent of the dimethyl sulphoxide concentration and a second phase where their rates are dependent on the concentration of dimethyl sulphoxide. Rama 25 cells do not become comitted to differentiate during this lag phase but increasing numbers of cells do so after this period. We suggest that the differentiation processes occur in two stages. The first stage, involving the inducer, commits Rama 25 cells to a new differentiated state. The second stage, involving the hormones, modulates the expression of different markers of this state. Both casein production and dome formation can be blocked by inhibitors of DNA synthesis and show reciprocal changes with the rates of cellular DNA synthesis. Thus, in its hormonal and DNA synthetic requirements for differentiation, Rama 25 cells appear to resemble some of the mammary epithelial cells of mature virgin rats.
The Interaction of Mammalian Medium‐Chain Hydrolase with Yeast Fatty Acid Synthetase
Authors: SLABAS Antoni R. , ORMESHER Jane , ROBERTS Peter A. , SIDEBOTTOM Christopher M. , TOMBS Michael P. , JEFFCOAT Roger , JAMES Anthony T.
Published: March 2005

The interaction of rat mammary gland medium‐chain thioesterase with yeast fatty acid synthetase has been investigated.

Medium‐chain thioesterase interacts with yeast fatty acid synthetase causing premature chain termination of the fatty acids synthesized from acetyl‐CoA and malonyl‐CoA. This effect is most marked under conditions of rate‐limiting malonyl‐CoA availability.

Immobilized yeast fatty acid synthetase specifically binds rat mammary gland medium‐chain thioesterase. This interaction has been used to purify medium‐chain thioesterase to near homogeneity from samples of rat mammary gland cytosol.

The stoichiometry of binding of medium‐chain thioesterase to yeast fatty acid synthetase has been investigated. Yeast fatty acid synthetase binds 5.7 ± 1 mol medium‐chain thioesterase/mol yeast fatty acid synthetase.

It is concluded that yeast fatty acid synthetase has a medium‐chain thioesterase binding site.