bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Reduction of Putidamonooxin by NADH–Putidamonooxin Oxidoreductase, Sodium Dithionite and Superoxide Radicals
Authors: BERNHARDT Frithjof‐Hans , KUTHAN Hartmut
Published: July 2010

Putidamonooxin, a conjugated iron‐sulfur protein, functions as the oxygen‐activating component within the 4‐methoxybenzoate monooxygenase from Pseudomonas putida. A[2Fe–2S] cluster and a mononuclear non‐heme iron together form the oxygen‐activating units within the active sites of putidamonooxin. In the present paper the reduction kinetics of oxidized putidamonooxin were studied in the absence and in the presence of a substrate and/or the mononuclear non‐heme iron. The reducing agenst used were (a) NADH plus the NADH–putidamonooxin oxidoreductase and (b) artificial reductants, i.e. O2 radicals and sodium dithionite in the presence or in the absence of the NADH‐putidamonooxin oxidoreductase. The reduction kinetics of cofactor‐iron‐depleted putidamonooxin revealed that the addition of a substrate strongly enhanced the rate of reduction of the [2Fe–2S] chromophores by the native reductase but significantly decreased the reduction by artificial reductants. These effects were even more pronounced when the mononuclear non‐heme iron was bound to putidamonooxin in addition to the substrate. The presence of the native reductase markedly enhanced the rate of reduction of native putidamonooxin by sodium dithionite, though not to the optimal rate as measured in the presence of both NADH and the NADH–putidamonooxin oxidoreductase.

The experimental findings are taken to show that the reduction of oxidized putidamonooxin in the absence and in the presence of its mononuclear non‐heme iron, when NADH–putidamonooxin oxidoreductase and artificial reductants are used as electron donors, may occur at two different sites, both of which are influenced by substrate binding. The most powerful reductant of putidamonooxin is the NADH–putidamonooxin oxidoreductase in its partially reduced state (which is characterized by its broad absorption band centered near 600 nm and its typical isotropic electron‐paramagnetic‐resonance signal with g= 2.0033), rather than in its fully reduced state.
The Role of Pyrrolo‐quinoline Semiquinone Forms in the Mechanism of Action of Methanol Dehydrogenase
Authors: DE BEER Ron , DUINE Johannis A. , FRANK Jzn , WESTERLING Jaap
Published: July 2010

On storage of methanol dehydrogenase of Hyphomicrobium X as a solution, the electron spin resonance (ESR) spectrum becomes more complicated. Ka‐band (35 GHz) spectroscopy reveals that a paramagnetic species with a lower g‐vale is formed from pyrrolo‐quinoline semiquinone (PQQH) during the ageing process. From the way in which this second paramagnetic species is formed and the absence of an effect of substrate, activator or both on fresh or aged enzyme, it follows that the complex ESR spectrum cannot be used as an argument to support the view that a three‐electron reduced form of PQQ exists in methanol dehydrogenase.

X‐band ESR spectroscopy of an enzyme solution reveals additional peaks at g= 2.03 and g= 2.01 only when the preparation contains O2 and is measured at liquid nitrogen temperature. The results point to the formation of a complex from PQQH and O2, most probably a peroxyl radical which only exists at low temperature.

Direct reduction of methanol dehydrogenase, as it is isolated, is possible. The enzyme form thus produced has the same properties as that obtained from reduction of oxidized methanol dehydrogenase with substrate, containing only a low amount of PQQH‐but not other free radical species. This substantiates the earlier conclusion that the isolated enzyme is in a half‐oxidized form.

A view on the mechanism of action of the enzyme is proposed which is quite opposite to the one that was recently advocated [Mincey et al. (1981) Biochemistry, 20, 7502–7509] since former and present results lead to the following conclusions: (a) there is no evidence for the involvement of a three‐electron reduced form of PQQ in the catalytic cycle of the enzyme; (b) enzyme molecules containing free radical species are not reduced by substrate; (c) the product is released before the enzyme is oxidized by electron acceptor.
UDPglucosyltransferase and Its Kinetic Fluorimetric Assay
Authors: VÄISÄNEN Matti V. T. , MACKENZIE Peter I. , HÄNNINEN Osmo O. P.
Published: July 2010

A rapid, Kinetic assay for UDPglucosyltransferase has been developed using 1‐naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1‐naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ.

Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP‐glucuronic acid, UDP‐N‐acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1‐naphthol and UDP‐glucose have also been determined.
Purification and Characterization of a Pig Intestinal α‐Limit Dextrinase
Authors: TARAVEL Francois R. , DATEMA Roelf , WOLOSZCZUK Wolfgang , MARSHALL J. John , WHELAN William J.
Published: July 2010

Mucosa from the duodenal and jejunal regions of pig small intestine was repeatedly freeze‐thaw treated to solubilize an enzyme preparation, enriched in maltase, glucoamylase and α‐limit dextrinase activities; isomaltase and sucrase remained essentially insoluble during the treatment.

Chromatographic procedures, including ion‐exchange, gel filtration and hydroxylapatite chromatography of the solubilized preparation, brought to homogeneity an α‐glucosidase active towards maltose, α‐limit dextrins and starch in decreasing order, with only a very weak capacity to hydrolyse α‐1,6‐linkages.

Michaelis constants and maximal velocities, as well as relative rates of hydrolysis of several substrates, including maltodextrins and α‐limit dextrins, were determined and served to characterize what seems to be a rather specific α‐1,4‐glucosidase.

The participation of this enzyme in the hydrolysis of α‐limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.
Selective Expression of Cloned Middle‐Repetitive Sequences in Nuclear RNA of Mouse Organs
Authors: KUROIWA Atsushi , OBINATA Masuo , NATORI Shunji
Published: July 2010

Mouse middle‐repetitive sequences have been cloned in the bacterial plasmid pBR322. The DNA sequences, expressed only in nuclei, were screened by filter hybridization using nuclear RNA as probes. Several clones were expressed predominantly in the nuclear RNA in certain types of organs. The copy numbers of typical middle‐repetitive sequence clones in genomic DNA were analyzed quantitatively and results showed that 103–104 copies exist per haploid DNA, indicating that they belong to a relatively abundant class of repeated sequences. Homology within the family of each middle‐repetitive sequence ranged from 81% to 94%.

The expression of cloned middle‐repetitive sequences was analyzed by liquid‐phase hybridization with nuclear RNAs from particular organs. The content of transcripts of the middle‐repetitive sequence in nuclear RNA of a particular organ was at least 1000 times higher than that in other organs. In steady‐state nuclear RNA these transcripts were calculated to amount to 102–104 copies per nucleus. Most of these transcripts seem to be retained in the nuclei and do not appear in the cytoplasm.
Fatty Acid in Lipopolysaccharides of Salmonella Species Grown at Low Temperature
Authors: WOLLENWEBER Horst‐Werner , SCHLECHT Siegfried , LÜDERITZ Otto , RIETSCHEL Ernst Theodor
Published: July 2010

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis‐Δ9‐16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid‐A portion of lipopolysaccharides, when the cells are grown at low temperature (12°C). It has recently been shown, that in S. minnesota R 595 grown at 37°C dodecanoic acid is linked to the 3‐hydroxyl group of an amide‐bound 3‐hydroxytetradecanoic acid. The present data suggest, that cis‐Δ9‐16:1 occupies the same position in lipid A (12°C).
Synthesis of Vimentin in a Reticulocyte Cell‐Free System Programmed by Poly(A)‐Rich RNA from Several Cell Lines and Rat Liver
Authors: McTAVISH Catharine F. , NELSON W. James , TRAUB Peter
Published: July 2010

Poly(A)‐rich RNA has been isolated from Ehrlich ascites tumour (EAT) cells and translated in a rabbit reticulocyte cell‐free system. The intermediate filament protein, vimentin, was found to be a major translation product. Fractionation of the poly(A)‐rich RNA by sucrose gradient centrifugation showed that the vimentin mRNA had a sedimentation coefficient of about 18 S corresponding to a molecular size of about 2000 nucleotides. This means that it must possess significant non‐coding regions. Vimentin synthesized in vitro was identical to native vimentin with regard to its precipitability with ammonium sulphate, extent of phosphorylation and susceptibility to digestion by the vimentin‐specific, Ca2+‐activated proteinase. Poly(A)‐rich RNA was also isolated from a number of tissue‐culture cells and rat liver, which contain varying amounts of vimentin in situ. It was found that the amount of vimentin synthesized by these RNA preparations in a rabbit reticulocyte cell‐free system is proportional to the amount of vimentin detectable in situ, suggesting that the amount of cellular vimentin may be controlled at the level of transcription.
Glycogen Synthase from Rabbit Skeletal Muscle; Effect of Insulin on the State of phosphorylation of the Seven Phosphoserine Residues in vivo
Authors: PARKER Peter J. , CAUDWELL F. Barry , COHEN Philip
Published: July 2010

Rabbits were starved for 24 h and injected with propranolol (2 mg/kg) with or without insulin (33 μg/kg) 15 min prior to sacrifice. In muscle extracts prepared from propranolol‐treated animals the activity ratio (± glucose 6‐phosphate) of glycogen synthase was 0.18 ± 0.02 and the Ka for glucose 6‐phosphate was 1.2 ± 0.1 mM. In (propranolol + insulin)‐treated animals the activity ratio was 0.35 ± 0.02 and the Ka for glucose 6‐phosphate was 0.60 ± 0.05 mM.

Glycogen synthase purified to homogeneity from propranolol‐treated animals contained 2.74 ± 0.09 mol phosphate/mol subunit, whereas this value was 2.33 ± 0.09 mol phosphate/mol subunit from (propranolol + insulin)‐treated animals.

The distribution of phosphate between the seven phosphorylation sites was elucidated. In (propranolol + insulin)‐treated animals there was a decrease of 0.4—0.45 mol phosphate/mol subunit in sites (3a + 3b + 3c), and no significant changes in any other site (1a, 1b, 2 and 5). The results demonstrate that dephosphorylation of sites (3a + 3b + 3c) is responsible for the activation of glycogen synthase observed after acute administration of insulin.

Neither protein phosphatase 1 nor protein phosphatase 2A, which account for virtually all the glycogen synthase phosphatase activity in skeletal muscle extracts, were able to mimic the specific dephosphorylation of sites (3a + 3b + 3c) produced by insulin. Both enzymes dephosphorylated site 2 and sites (3a + 3b + 3c) in vitro at comparable rates. The action of insulin may therefore involve a decrease in the activity of glycogen synthase kinase 3 and the regulation of this enzyme is discussed.

The failure of insulin to produce a significant dephosphorylation of the sites phosphorylated by cyclic‐AMP‐dependent protein kinase (1a, 1b and 2) demonstrates that inhibition of this protein kinase does not underlie the activation of glycogen synthase by this hormone. This conclusion is supported by measurements of the phosphorylation states of inhibitor 1, phosphorylase kinase and phosphorylase.

Neither 24‐h starvation, nor alloxon‐induced diabetes, produced significant changes in the kinetic properties or phosphorylation state of glycogen synthase. Possible reasons for these findings are discussed.
Purification and characterization of the 90‐kDa heat‐shock protein from mammalian tissues
Authors: YONEZAWA Naoto , NISHIDA Eisuke , SAKAI Hikoichi , KOYASU Shigeo , MATSUZAKI Fumio , IIDA Kazuko , YAHARA Ichiro
Published: July 2010

The 90‐kDa heat‐shock protein (HSP90) has been purified from mammalian tissues, mouse liver and porcine brain, with a good yield by a new method involving hydrophobic chromatography. Mouse liver HSP90 and porcine brain HSP90 were compared with mouse lymphoma HSP90 which was purified from T lymphoma cell line. L5178Y, by a modification of the previously reported method. These three‐HSP90s were indistinguishable from one another in amino acid composition, one‐dimensional peptide mapping, elution pattern of proteolytic fragments (trypsin‐ or V8‐protease‐cleaved) in reverse‐phase high‐performance liquid chromatography, reactivity with the antibody against mouse T lymphoma HSP90 and the ability to bind to F‐actin. The amino acid sequences of three portions (total 47 amino acid residues) of lymphoma HSP90 were determined and they were homologous to those of the corresponding portions of Drosophila HSP83A and yeast HSP90. These results suggest that HSP90 is a highly conserved protein during evolution.
Primary and secondary structure of the 18S ribosomal RNA of the bird spider Eurypelma californica and evolutionary relationships among eukaryotic phyla
Authors: HENDRIKS Lydia , van BROECKHOVEN Christine , VANDENBERGHE Antoon , van de PEER Yves , de WACHTER Rupert
Published: July 2010

The primary structure of the 185 rRNA of the bird spider Eurypelma californica has been determined in the framework of a study of metazoan phylogeny on the basis of ribosomal RNA structure. A secondary‐structure model was derived by comparison of the sequence with that of 43 other eukaryotic small‐ribosomal‐subunit RNA sequences presently available. This comparison allows a rather detailed secondary‐structure pattern to be postulated for a eukaryote‐specific area of highly variable sequence and length for which no consensus model has hitherto been attained. A dendrogram, reflecting evolutionary relationships among the 40 eukaryotic species of known 18S rRNA structure, was constructed by a matrix method selecting the best‐fitting tree on the basis of a least‐squares criterion. The tree shows an early divergence of a microsporidium, an euglenoid, kinetoplastids and a slime mold. Among the remaining species, two main clusters are distinguishable, one comprising the Ciliata, the other comprising Metazoa, green plants, fungi and several protists. Among the Metazoa, the three phyla presently investigated, viz. Chordata. Arthropoda and Nemathelminthes, are distinguishable as three separate lines of descent.
Molecular cloning of murine p68, a Ca2+‐binding protein of the lipocortin family
Authors: MOSS Stephen E. , CROMPTON Mark R. , CRUMPTON Michael J.
Published: July 2010

A plasmid cDNA library prepared from a T‐lymphocyte clone of murine strain B10.A origin was screened by cross‐species DNA hybridisation using a partial human p68 cDNA clone, identified as containing coding sequences for previously determined amino‐acid sequences. The longest p68 cDNA insert from this library and a full‐length cDNA insert from a second similar library were fully sequenced. A comparison of the derived amino‐acid sequence with that of human p68 revealed extensive homology (95% overall). Homology at the nucleotide level was 89% in the open reading frame and 85% and 50% in the 5′ (33 nucleotides) and 3′ (347 nucleotides) non‐coding regions respectively. Eight segments of internal homology were observed, each containing a highly conserved consensus region of 17 amino acids correlating with that described for several membrane associated calcium‐binding proteins [Geisow, M. J., Fritsche, U., Hexham, J. M. & Johnson, T. (1986) Nature (Lond.) 320, 636–638]. These results provide further evidence that p68 is a member of the same gene family as p32, p36 and lipocortin I and demonstrate an unusually high level of inter‐species sequence conservation of p68 between mouse and human.
Isolation and sequence analysis of the fatty acid synthetase FAS2 gene from Penicillium patulum
Authors: WIESNER Peter , BECK Joachim , BECK Karl‐Friedrich , RIPKA Sabine , MÜLLER Gerhard , LÜCKE Silvia , SCHWEIZER Eckhart
Published: July 2010

The fatty acid synthetase complex of Penicillium patulum was isolated and shown to be structurally similar to other known fungal fatty acid synthetases. It is composed of two subunit, α and β, each with a molecular mass of about 200 kDa. P. patulum genomic and cDNA libraries were constructed in λgt11 and EMBL3 vectors. From these libraries, the P. patulum FAS2 gene together with its flanking DNA was isolated. The cloned genomic DNA was sequenced over a length of 6357 base pairs. The coding sequence of fatty acid synthetase subunit α, being 5571 nucleotides long, was identified within this DNA segment. The FAS2 gene is a mosaic of three exons (514, 4949 and 108 base pairs) and two introns, each of 54 base pairs in length. Both introns were absent in the corresponding cDNA sequences. Like other fungal introns both contain an internal CTAAC sequence. located 10 base pairs upstream of their 3′‐exon/intron boundaries. In addition, they have, at their ends, the GCTAAGT and TAG consensus sequences characteristic of all eucaryotic introns. Furthermore, two pairs of direct repeats, of as yet unknown significance, were found in the two P. patulum introns. The P. patulum FAS2 gene encodes a protein of 1857 amino acids and 204.5 kDa molecular mass. It is 90 nucleotides shorter than the corresponding S. cerevisiae gene. In both organisms, the FAS2 genes and their products exhibit a high degree of overall sequence similarity at both the DNA (63%) and protein (68%) levels. Therefore, the fatty acid synthetase α subunits of P. patulum and S. cerevisiae obviously contain the same catalytic domains in an identical sequential order.
Differential inhibition of various deoxyribonucleic acid polymerases by Evans blue and aurintricarboxylic acid
Authors: NAKANE Hideo , BALZARINI Jan , de CLERCQ Erik , ONO Katsuhiko
Published: July 2010

The inhibitory effects of two anionic compounds, Evans blue and aurintricarboxylic acid (ATA), on various kinds of polynucleotide‐synthesizing enzymes were examined. Under the assay conditions, optimized for each enzyme species, both these compounds strongly inhibited the activities of the purified human DNA polymerases α, β, γ, and DNA primase as well as those of DNA polymerase I and RNA polymerase from Escherichia coli and Rauscher leukemia virus reverse transcriptase. ATA was particularly effective in inhibiting retroviral reverse transcriptase and cellular DNA polymerase α. Evans blue, which is a structural analogue of suramin, exerted its inhibitory action largely by competing with the template · primer for the same binding site of the enzyme. On the other hand, ATA inhibited most, if not all, of these enzyme activities noncompetitively with respect to either the template · primers or nucleoside 5′ ‐triphosphate substrates. The inhibition constants for ATA were, in general, smaller than those for Evans blue.
Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose‐containing polysaccharide with a chondroitin backbone
Authors: RODRIGUEZ Maria‐Luisa , JANN Barbara , JANN Klaus
Published: July 2010

The chemical structure of the K4‐specific capsular polysaccharide (K4 antigen) of Escherichia coli O5:K4:H4 was elucidated by composition, carboxyl reduction periodate oxidation methylation nuclear‐magnetic‐resonance spectroscopy and enzymatic cleavage. The polysaccharide consists of a backbone with the structure → 3)‐β‐D‐glucuronyl‐(1,4)‐β‐D‐N‐acetylgalactosaminyl(1‐ to which β‐fructofuranose is linked at C‐3 of glucuronic acid. Mild acid hydrolysis liberated fructose and converted the K4 antigen into a polysaccharide which has the same structure as chondroitin. The defructosylated polysaccharide was a substrate for hyaluronidase and chondroitinase. The serological reactivity of the K4 polysaccharide was markedly reduced after defructosylation.
Phosphatases and phosphodiesterases interfere with 1,3‐β‐D‐glucan synthase activity in pea epicotyl membrane preparations
Authors: SAUGY Martial , FARKAS Vladimir , MACLACHLAN Gordon
Published: July 2010

Soluble and membrane‐bound phosphatase and phosphodiesterase activities are present in preparations of 1,3‐β‐D‐glucan synthase from pea epicotyls. UDP‐glucose phosphodiesterase and non‐specific alkaline phosphatase could be partially inhibited by N‐ethylmaleimide or iodoacetamide and partially removed from membranes by washing. Such treatments helped to prolong 1,3‐β‐glucan synthase activity. Nevertheless, the 1,3‐β‐D‐glucan synthase activity in washed membranes still gradually decreased during incubation in buffer at 30°C. The rate of decay was reduced by adding more specific phosphatase inhibitors, e.g. molybdate, vanadate or fluoride, or by addition of nucleotides, and much of the loss of 1,3‐β‐D‐glucan synthase activity during preincubation could be restored by addition of phosphatidylethanolamine to the assay mixtures. It is concluded that membrane phospholipid is an essential part of the environment of 1,3‐β‐glucan synthase and must be maintained intact in order for the enzyme to remain fully active.
Proton‐NMR studies show that the Thr‐102 mutant of yeast iso‐1‐cytochrome c is a typical member of the eukaryotic cytochrome c family
Authors: PIELAK Gary J. , BOYD Jonathan , MOORE Geoffrey R. , WILLIAMS Robert J. P.
Published: July 2010

The Thr‐102 mutant of yeast iso‐1‐cytochrome c is a useful system for the study of structure‐function relationships in this important class of electron transfer proteins, but little is known about its structure. Furthermore, few assignments of individual amino acid residues in yeast iso‐1‐cytochrome c have been made by proton NMR. Here we report assignments for nearly half of the amino acids in the reduced Thr‐102 mutant of yeast iso‐1‐cytochrome c. We also report assignments for the oxidized Thr‐102 mutant. While the crystal structure of the reduced iso‐1‐cytochrome c (N. B. not the Thr‐102 mutant) has been reported, there is currently little structural information concerning its solution structure and none concerning the oxidized protein. There is also no information concerning the structure of either oxidation state of the Thr‐102 mutant. Comparison of the chemical shift and NOE data for the reduced Thr‐102 mutant and comparison of paramagnetic shifts for analogous residues between this mutant and horse‐heart and tuna cytochromes c reveal that both the basic fold of Thr‐102 yeast iso‐1‐cytochrome c and the region around the site of the mutation are the same as those found in the latter two proteins. It is concluded that the results from structure function studies using the Thr‐102 mutant will be applicable to eukaryotic cytochrome c in general. This knowledge allows us to proceed to a description of some mutants of yeast iso‐1‐cytochrome c in the next paper.
Two‐dimensional NMR as a probe of structural similarity applied to mutants of cytochrome c
Authors: PIELAK Gary J. , ATKINSON R. Andrew , BOYD Jonathan , WILLIAMS Robert J. P.
Published: July 2010

Using site‐directed mutagenesis, it is possible to prepare many mutants of a protein in a short time, and to uncover differences in function. To understand the changes in function, it is essential to understand the effect(s) of the mutation in terms of structural and dynamic changes. It is particularly important to establish a rapid method for comparing the structure of the mutants with that of the wild‐type protein. We propose that a combination of overlayed and difference two‐dimensional NOE spectra between the wild‐type and mutant protein provide a rapid method for determination of structural similarity. The observation of differences other than those due directly to the field effects of the exchanged side chain allow both local and distant conformational changes to be assessed. Here we compare NOESY spectra from a mutant of yeast iso‐1‐ferrocytochrome c in which the invariant residue Phe‐82 has been changed to a Tyr. We conclude that NMR can show subtle changes in protein structure. Specifically, we show the change must involve the reorientation of the side chain of Leu‐85 which is proximal to the mutation. The dynamics of the aromatic side chain at position 82 are shown not to give rise to measurable differences between the wild‐type and mutant protein. Structural changes are not propagated to a measurable degree in other parts of the protein.
Localization of the synthesis of very‐long‐chain fatty acid in mitochondria from Saccharomyces cerevisiae
Authors: BESSOULE Jean‐Jacques , LESSIRE René , RIGOULET Michel , GUERIN Bernard , CASSAGNE Claude
Published: July 2010

The localization of the mitochondrial elongation activities (‘elongases’) from Saccharomyces cerevisiae has been investigated. It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very‐long‐chain fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane‐bound enzymes. The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker. A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria.
The effect of inorganic pyrophosphate on the activity and Pi‐binding properties of mitochondrial F1‐ATPase
Authors: KALASHNIKOVA Tatyana Yu. , MILGROM Yakov M. , MURATALIEV Marat B.
Published: July 2010

Interaction of F1‐ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1‐ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady‐state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP‐dependent reactivation of an inactive complex formed by F1‐ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1‐ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non‐preincubated enzyme, PPi stimulates the binding of Pi to F1‐ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high‐affinity Pi binding. It follows from the results obtained that PPi interacts with the non‐catalytic site(s) of F1‐ATPase.
Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii
Authors: BREIDENBACH Eric , JENNI Erika , BOSCHETTI Arminio
Published: July 2010

Chloroplasts contain thylakoid‐bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid‐bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32‐kDa herbicide‐binding membrane protein and the soluble large subunit of the ribulose‐1,5‐bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75–90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10‐fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA‐binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light‐induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.
Enzymatic methyl esterification of synthetic tripeptides: structural requirements of the peptide substrate
Authors: GALLETTI Patrizia , INGROSSO Diego , MANNA Caterina , SICA Filomena , CAPASSO Sante , PUCCI Piero , MARINO Gennaro
Published: July 2010

Eukaryotic protein carboxyl methyltransferase catalyzes a two‐substrates reaction in which the methyl group of S‐adenosylmethionine is transferred to the free carboxyl group of D‐aspartyl and L‐isoaspartyl‐containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595–602], while very little is know concerning the structural requirements of the protein substrate.

In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl‐accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme‐substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11 503–11 511]; (2) the isoaspartyl residue is not recognized unless its α‐amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D‐aspartyl and D‐isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed.

This paper also reports the first application of fast‐atom‐bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methylesterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.
Display of Expression Products of cDNA Libraries on Phage Surfaces
Authors: Crameri Reto , Jaussi Rolf , Menz Günter , Blaser Kurt
Published: July 2010

Techniques for cloning cDNAs from bacteriophage libraries immobilised on solid supports are well established. However, these techniques do not allow selective enrichment of clones expressing proteins of interest. Screening of cDNA libraries would be simplified if the proteins encoded by cDNAs could be expressed on the surface of phage. Phage carrying genes encoding proteins for which a ligand is available can be selected directly by affinity interaction [Crameri, R. & Suter, M. (1993) Gene (Amst.) 137, 69–75]. The expression products from a cDNA library from Aspergillus fumigatus have been displayed on the surface of the filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA‐encoded proteins to the genetic information required for their production, achieved by exploiting the high‐affinity interaction of the Jun and Fos leucine zippers, allows screening of up to 1 × 1010 independent clones in 50‐μl aliquots applied to a well of a microtiter plate coated with the ligand. Phage displaying IgE‐binding proteins were selectively enriched 105–106‐fold over non‐specific phage after six rounds of growth and selection. The apparent molecular mass of the proteins selected from the cDNA library was in the range 20–40 kDa. Restriction enzyme analysis and preliminary sequence determination of 12 selected inserts revealed different sequences. The ability of the proteins to bind to human serum IgE was corroborated by enzyme‐linked immunosorbent assay and by Western‐blot analysis. The developed cloning strategy allows isolation of cDNAs encoding proteins for which a ligand is available and circumvents immobilisation of the libraries on solid‐phase supports which hamper selective enrichment of clones expressing the desired protein.
Purification and Characterization of the Poly(Hydroxyalkanoic Acid) Synthase from Chromatium vinosum and Localization of the Enzyme at the Surface of Poly(Hydroxyalkanoic Acid) Granules
Authors: Liebergesell Matthias , Sonomoto Kenji , Madkour Mohamed , Mayer Frank , Steinbüchel Alexander
Published: July 2010

A recombinant strain of Escherichia coli, which overexpressed phaC and phaE from Chromatium vinosum, was used to isolate poly(3‐hydroxyalkanoic acid) synthase. The isolation was performed by a two‐step procedure including chromatography on DEAE‐Sephacel and Procion Blue H‐ERD. The poly(3‐hydroxyalkanoic acid) synthase consisted of two different kinds of subunit (PhaC, M, 39500 and PhaE, Mr 40500). PhaC was separated from the poly(3‐hydroxyalkanoic acid) synthase complex by chromatography on phenyl‐Sepharose; PhaE was enriched by solubilization of protein inclusion bodies. The stoichiometry of PhaC and PhaE in the enzyme complex was not determined. The poly(3‐hydroxyalkanoic acid) synthase (PhaEC) exhibited a native relative molecular mass of Mr 400000 and most probably consists of ten subunits. The Kmvalue of the enzyme for D(–)‐3‐hydroxybutyryl‐CoA was 0.063 mM. The enzyme synthesized poly(3‐hydroxybutyric acid) in vitro from D(–)‐3‐hydroxybutyryl‐CoA or, together with propionyl‐CoA transferase in a coupled enzyme reaction, synthesized the same product from acetyl‐CoA plus d(–)‐3‐hydroxybutyric acid. Antibodies were raised against both subunits of the poly(3‐hydroxyalkanoic acid) synthase. By immunoelectron microscopy, the poly(3‐hydroxyalkanoic acid) synthase was localized within the cytoplasm in cells of C. vinosum grown under non‐storage conditions. In cells grown under poly(3‐hydroxybutyric acid) storage conditions, the enzyme was observed to be located at the surface of the poly(3‐hydroxybutyric acid) granules. Immunoblots with anti‐PhaC, anti‐PhaE IgG and crude extract proteins indicated that poly (3‐hydroxyalkanoic acid) synthases with partial sequence similarities are widespread among purple sulphur bacteria.
Regulation of 70‐kDa Heat‐Shock‐Protein ATPase Activity and Substrate Binding by Human DnaJ‐Like Proteins, HSJ1a and HSJ1b
Authors: Cheetham Michael E. , Jackson Antony P. , Anderton Brian H.
Published: July 2010

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70‐amino‐acid J domain. Escherichia coli DnaJ interacts with the 70‐kDa heat‐shock protein (DnaK), in vitro, to stimulate the 70‐kDa heat‐shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ‐like proteins with the 70‐kDa heat‐shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone‐specific DnaJ‐like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine‐rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70‐kDa heat‐shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70‐kDa heat‐shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected.

HSJ1 proteins appear to regulate the affinity of the 70‐kDa constitutive heat‐shock protein for the permanently unfolded substrate, carboxymethylated α‐lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664–666] has suggested that substrate release by 70‐kDa heat‐shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70‐kDa constitutive heat‐shock protein/carboxymethylated α‐lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70‐kDa constitutive heat‐shock protein that can mimic the effect mediated by K+ and therefore modulate 70‐kDa heat‐shock protein substrate release by another mechanism rather than merely stimulating the 70‐kDa heat‐shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70‐kDa heat‐shock‐protein substrate binding will be common to all proteins that contain a J domain.
Interleukin‐1‐Induced Prostaglandin E2 Biosynthesis in Human Synovial Cells Involves the Activation of Cytosolic Phospholipase A2 and Cyclooxygenase‐2
Authors: Angel Juana , Berenbaum Francis , Le Denmat Corinne , Nevalainen Timo , Masliah Joëlle , Fournier Catherine
Published: July 2010

Treatment of human synovial cells with interleukin‐1 (IL‐1) results in a large increase in the production of prostaglandin E, (PGE2), a function in which the activation of phospholipase A2 (PLA2) is a key step. In order to identify the enzymes that are linked to IL‐1‐mediated arachidonate availability and subsequent PGE2 production, we have investigated the changes in gene expression of the 85‐kDa cytosolic PLA2 (cPLA2), the 14‐kDa secretory PLA2 (sPLA2) and the two forms of cyclooxygenase in human synoviocytes after stimulation with recombinant IL‐1β. Northern‐blot analysis revealed that both cPLA2 and cyclooxygenase‐2 mRNA were progressively upregulated upon exposure to IL‐1 for 5 hours and the glucocorticoid, dexamethasone, blocked the increased expression of these two genes. In contrast, IL‐1‐induced sPLA2 gene expression determined in the same cell samples was weak and most often rapid, and dexamethasone further stimulated it. In addition, IL‐1 did not modify the levels of the constitutive cyclooxygenase‐1. The cPLA2 and cyclooxygenase‐2 enzymic activities are dependent upon de novo synthesis of mRNA and protein, since they were inhibited by actinomycin D and cycloheximide. Our data suggest that the IL‐1‐induced production of PGE2 in human synoviocytes can be attributed to the stimulation of both cPLA2 and cyclooxygenase‐2. These enzymes may represent appropriate targets for selective blockade of prostanoid production in the inflammed joints.
Comparative Analysis of Ribosome‐Associated Adenosinetriphosphatase (ATPase) from Pig Liver and the ATPase of Elongation Factor 3 from Saccharomyces cerevisiae
Authors: Kovalchuke Oleh , Chakraburtty Kalpana
Published: July 2010

Elongation factor 3 (EF‐3) is a unique and essential requirement of the fungal translational machinery. Non‐fungal organisms do not have and do not require a soluble form of the third elongation factor for translation. To test whether non‐fungal organisms have a direct analog of EF‐3 incorporated in the structure of the ribosomes, a comparison of EF‐3 adenosinetriphosphatase (ATPase) with ATPases associated with pig liver ribosomes was carried out. EF‐3 function depends on ATP (GTP) hydrolysis. The hydrolytic activity of EF‐3 is enhanced by two orders of magnitude by yeast ribosomes due to an increase in the turnover rate of EF‐3. The nucleotide hydrolytic activity of EF‐3 is significantly constrained by the binding of aminoacylated tRNAphe to poly(U)‐programmed ribosomes. The translational inhibitors – neomycin and α‐sarcin suppress the ATPase activity of EF‐3. These results reflect a direct correlation between EF‐3 ATPase and the functional state of the ribosome. Four lines of evidence indicate that yeast EF‐3 ATPase is functionally distinct from pig liver ribosome associated ATPases. The kinetic parameters of ATPases from these two sources are different. Poly(U) and tRNA have no effect on the ATPase activity associated with the pig liver ribosomes. The latter activity is unaffected by the translational inhibitors neomycin and α‐sarcin. The translational activity of pig liver ribosomes is not influenced by ATP, ADP or adenosine 5′‐[β, γ‐imido]triphosphate. In an in vitro system, one can demonstrate a small but consistent stimulatory effect of yeast EF‐3 on polyphenylalanine synthesis by pig liver ribosomes only when EF‐1α is present at a limited concentration. The EF‐3 effect disappears when EF‐1α is added in a stoichiometric amount to the pig liver ribosomes. This result is in contrast to the yeast system where the ribosomes are completely dependent on EF‐3 at all concentrations of EF‐1α.
Expression of the Mau Genes Involved in Methylamine Metabolism in Paracoccus denitrificans is Under Control of a LysR‐type Transcriptional Activator
Authors: Van Spanning Rob J. M. , van der Palen Carol J. N. M. , Slotboom Dirk‐Jan , Reijnders Willem N. M. , Stouthamer Adriaan H. , Duine Johannis A.
Published: July 2010

Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate‐induction mechanism as well as by a catabolite‐repression‐like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR‐type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane‐embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.
31P‐NMR Studies of NADPH, NADP+ and the Complex of NADPH and Methotrexate with Lactobacillus casei Dihydrofolate Reductase in the Solid State
Authors: Gerothanassis Ioannis P. , Barrie Patrick J. , Birdsall Berry , Feeney James
Published: July 2010

31P‐NMR spectra on solid samples of NADP+, NADPH and NADPH bound to Lactobacillus casei dihydrofolate reductase have been recorded using the techniques of cross polarisation, magic angle spinning and high power proton decoupling. The isotropic chemical shifts, the principal components of the shielding tensors and the asymmetry parameters for the 31P nuclei in the 2′‐phosphate and pyrophosphate groups have been measured. The isotropic shifts show similar trends to the chemical shifts measured in solution. The isotropic shifts and the shielding tensors for the dianionic and monoanionic states of the 2′‐phosphate group have been determined and the presence of both ionisation states has been detected in a solid sample of the lyophilised complex of L. casei dihydrofolate reductase with NADPH and methotrexate. This contrasts with the behaviour in solution, where only the dianionic form is bound to the enzyme. The signals from the two pyrophosphates 31P nuclei in bound NADPH were resolved and identified. The asymmetry parameters in the different ionisation states and the orientations of the shielding tensors within the molecular framework are considered in the context of previous 31P studies on phosphate‐containing compounds.
Regulation of Globin Gene Expression in Erythroid Cells
Authors: Orkin Stuart H.
Published: July 2010

Study of globin gene regulation has served as a useful paradigm for cell‐specific and developmental control of transcription in higher eukaryotic cells. Recent work directed toward the identification and characterization of the cis‐regulatory elements and transcription factors important for both aspects of control is reviewed. Particular emphasis is placed on the organization and function of globin locus control regions, mechanisms of switching of globin gene expression during development, and functions of the major erythroid‐specific nuclear regulatory proteins.
Identification of a Transcriptional Regulatory Factor for Human Aromatase Cytochrome P450 Gene Expression as Nuclear Factor Interleukin‐6 (NF‐IL6), a Member of the CCAAT/Enhancer‐Binding Protein Family
Authors: Toda Katsumi , Akira Shizuo , Kishimoto Tadamitsu , Sasaki Hiroshi , Hashimoto Kozo , Yamamoto Yasutake , Sagara Yusuke , Shizuta Yutaka
Published: July 2010

Human aromatase cytochrome P450 catalyzes the ultimate reaction in the estrogen biosynthetic pathway by coupling with another enzyme, NADPH‐cytochrome P450 reductase, in the endoplasmic reticulum. The expression of the gene encoding the enzyme (CYP19) is regulated, in part, by tissue‐specific promoters through the use of alternative‐splicing mechanisms. Recently, we have localized a transcriptional activating element at positions –2141 to –2115 relative to the major cap site of the gene, by transient expression analyses in human BeWo choriocarcinoma cells using the bacterial chloramphenicol acetytransferase reporter gene ligated with CYP19 promoter sequences which regulate expression in this tissue. Here, we report the isolation of a cDNA encoding a DNA‐binding protein which binds specifically to the regulatory element. The deduced amino‐acid sequence of the insert is identical to that corresponding to the DNA‐binding domain and the dimerization domain of a transcription factor, nuclear factor interleukin‐6 (NF‐IL6), a member of the CCAAT/enhancer‐binding protein (C/EBP) family. Studies using specific antibodies against members of the C/EBP family demonstrate that NF‐IL6 is the major nuclear factor binding to the regulatory element in BeWo cells; nevertheless, C/EBP α also seems to be involved. Disruption of the NF‐IL6‐binding site within the regulatory element resulted in the disappearance of the transcriptional enhancing activity of the element, indicating that NF‐IL6 is at least one of the nuclear factor(s) which enhances transcription through binding to the cis‐acting element. These results indicate the intrinsic importance of NF‐IL6 in the transcriptional regulation of CYP19 expression.
Chaperone‐Like Activity of Protein Disulfide‐Isomerase in the Refolding Of Rhodanese
Authors: Song Jiu‐li , Wang Chih‐chen
Published: July 2010

Protein disulfide‐isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine‐hydrochloride‐denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above‐mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515–1517] that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperone‐like activity of PDI acting on rhodanese is independent of its disulfide‐isomerase activity.