bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence and expression of the gene encoding the respiratory nitrous‐oxide reductase from Paracoccus denitrificans
Authors: HOEREN Frank U. , BERKS Ben C. , FERGUSON Stuart J. , McCARTHY John E. G. 
Published: March 2005

The structural gene for the respiratory nitrous‐oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo‐protein) using an expression vector in Escherichia coli.Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous‐oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N‐terminal sequencing of the mature nitrous‐oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous‐oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous‐oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C‐terminal region of the nitrous‐oxide‐reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities.Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position –21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position –126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans.Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species.An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous‐oxide reductase and which will be of general value in other studies of this organism.
Kinetic studies of pea carbonic anhydrase
Authors: JOHANSSON Inga‐Maj , FORSMAN Cecilia 
Published: March 2005

Chloroplast carbonic anhydrase from Pisum sativum has been isolated. The kinetic properties of the enzyme have been studied and comparisons to the well characterised human carbonic anhydrase II made. Pea carbonic anhydrase was found to be dependent on a reducing agent in order to retain the catalytic activity. Oxidised, inactive, enzyme could be activated by the addition of a SH‐agent. However, such activation gave only 60% of the activity of an enzyme kept in a reduced state all the time. The kinetics of CO2 hydration show an increase in Kcat as well as in kcat/Km with pH, but the pH profile does not follow a simple titration curve. The pH dependence is more complicated and it seems as if there are several titratable groups affecting the activity. At pH 9 we obtain a turnover number of 4x105 s−1 and a Kcat/Km value of 1.8x108 M−1 s−1 with reference to the subunit. We also find that the enzyme needs high concentrations of buffer to work at a maximal rate. Apparent Km values with respect to the total buffer concentration are found between 52–185 mM at neutral and high pH. At low pH the situation is complex with deviations from Michaelis‐Menten kinetics.Chloroplast carbonic anhydrase from higher plants have been reported to have primary structures that are completely different from the enzyme from animals. In addition, we find the circular dichroic spectrum of pea carbonic anhydrase to be well distinguished from that of human carbonic anhydrase II. Despite those structural differences the kinetic parameters indicate that pea carbonic anhydrase is equally efficient as human carbonic anhydrase II in catalysing the hydration of CO2. However, the mechanism for proton transfer from the active site to the surrounding medium seems to differ between the two enzymes.
The structure of the carbohydrate backbone of the core‐lipid‐A region of the lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1)
Authors: VINOGRADOV Evgeny V. , STUIKE‐PRILL Rainer , BOCK Klaus , HOLST Otto , BRADE Helmut 
Published: March 2005

Lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1) was de‐O‐acylated, dephosphorylated, reduced, de‐N‐acylated, N‐acetylated, and the products were separated by high‐performance anion‐exchange chromatography (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN‐disaccharide lipid A backbone. Its structure was established by compositional and methylation analyses, and extensive NMR investigations including 1H, 1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY), as well as heteronuclear 13C, 1H COSY. In another reaction sequence the lipopolysaccharide was hydrolysed with dilute acetic acid and reduced with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. A major product, 2, was isolated and investigated by the same methods as described for the decasaccharide 1. The following structures are proposed for compounds 1 and 2:

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where R is α‐Kdop‐(2–6)‐β‐D‐GlcNAcp‐(1–6)‐D‐GlcNAcol in 1 and 4,8‐anhydro‐Kdool in 2, and Hep is L‐glycero‐D‐manno‐heptose.In lipopolysaccharide, the terminal residue of α‐D‐glucosamine possessed a free amino group, as proved by deamination with nitrous acid and the 1H‐NMR spectrum of de‐O‐acylated lipopolysaccharide.The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simulations combined with restrained calculations of side chains based on experimentally determined proton‐coupling constants. These calculations, confirmed by NOE data, displayed several long‐range interactions, which resulted in a well‐defined three‐dimensional structure of the core oligosaccharide.
The actin/actin interactions involving the N‐terminus of the DNase‐I‐binding loop are crucial for stabilization of the actin filament
Authors: KHAITLINA Sofia Y. , MORACZEWSKA Joanna , STRZELECKA‐GOŁASZEWSKA Hanna 
Published: March 2005

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I. M., Pershina, V. P., Synakevich, I. G., Turoverov, K. K. & Usmanova, A. M. (1991) FEBS Lett. 279, 49–51]. The resulting C‐terminal and N‐terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+. The protease‐treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease‐treated actin to inhibit the DNase I activity.The critical concentration for KCl‐induced polymerization of ECP‐treated ATP–Mg–G‐actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisn, and 36‐fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP‐treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl‐labelled actins and chemical cross‐linking with N,N′‐1,2‐phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP‐treated actin and those of intact as well as subtilisin‐treated actin. Moreover, the filaments of ECP‐treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F‐actin [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44–49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.
The complex between ribonuclease T1 and 3′GMP suggests geometry of enzymic reaction path
Authors: HEYDENREICH Andrea , KOELLNER Gertraud , CHOE Hui‐Woog , CORDES Frank , KISKER Caroline , SCHINDELIN Hermann , ADAMIAK Ryszard , HAHN Ulrich , SAENGER Wolfram 
Published: March 2005

The crystal structure of the complex between ribonuclease T1 and 3′GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in‐line process, (c) His40 NɛH+ activates O2′‐H, (d) Glu58 carboxylate acts as base and His92 NɛH+ as acid in a general acid‐base catalysis.The crystals have the monoclinic space group P21, a= 4.968 nm, b= 4.833 nm, c= 4.048 nm, β= 90.62° with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R= 15.3% with 11338 data ≥1σ(F°) in the resolution range 1.0–0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3′GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4′‐exo puckering, the ribose phosphate is extended with torsion angle ɛ in trans. The O2′‐H group is activated by accepting and donating hydrogen bonds from His40 NɛH+ and to Glu58 Oɛ1; the phosphate is hydrogen bonded to Glu58 Oɛ2H, Arg77 NɛH+ and Nη2H+, Tyr38 OηH, His92 NɛH+. The conformation of ribose phosphate is such that O2′ is at a distance of 0.31 nm from phosphorus, and opposite the P‐OP3 bond which accepts a hydrogen bond from His92 NɛH+; we infer from a model building study that this bond is equivalent to the scissile P‐O5′ in a substrate GpN.
Monodehydroascorbate reductase activity in the surface membrane of leukemic cells
Authors: SCHWEINZER Esther , GOLDENBERG Hans 
Published: March 2005

A transmembrane monodehydroascorbate reductase activity with a high affinity in the subpicomolar concentration range of the free radical can be measured at the surface of erythroleukemic cells using a ferricyanide‐driven redox cycle. The activity is dependent on the membrane potential and can therefore only be found in intact cells. It is independent of the glutathione content of the cells. Thenoyltrifluoroacetone is an efficient inhibitor of the activity, whereas ouabain, monensin and tetraethylammonium show no effect.Cells are able to generate ascorbate from dehydroascorbic acid. This explains why both forms of vitamin C show practically the same affinity for the redox cycle but why it does not drive the redox cycle by itself because it is much slower and is not inhibited by thenoyltrifluoroacetone.The reductase activity is independent of the degree of differentiation of the leukemic cells.
The Ets family of transcription factors
Authors: WASYLYK Bohdan , HAHN Soonjung L. , GIOVANE Antoine 
Published: March 2005

Interest in the Ets proteins has grown enormously over the last decade. The v‐ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a ceilular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so‐called ‘ets domain’. The ets domain has been shown to be a DNA‐binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein–protein interactions with co‐factors that help determine its biological activity. Many of the Ets‐related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti‐oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets‐like proteins may have other functions, such as in DNA replication and a general role in transcription activation.Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T‐cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors, G‐proteins, kinases and transcription factors. We will discuss how several Ets‐related proteins fit into this scheme, and how their activity is regulated both post‐and pre‐translationally. Loss of normal control is often associated with conversion to an onco‐protein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene‐related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets‐family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness.
The use of lac‐type promoters in control analysis
Authors: JENSEN Peter Ruhdal , WESTERHOFF Hans V. , MICHELSEN Ole 
Published: March 2005

For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady‐state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild‐type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lac Yam mutation (eliminating lactose permease, the transport system for the inducer isopropyl‐thio‐β‐D‐galactoside) is introduced.The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+‐ATPase, and found that the expression of this enzyme could be modulated between non‐detectable levels and up to five times the wild‐type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild‐type level. We show that the introduction of a lac Y mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl‐thio‐β‐D‐galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.
Further Characterisation of Forms of Haemosiderin in Iron‐Overloaded Tissues
Authors: Ward Roberta J. , Ramsey Michael , Dickson Dominic P. E. , Hunt Collette , Douglas Trevor , Mann Stephen , Aouad Fouad , Peters Timothy J. , Crichton Robert R. 
Published: March 2005

The biochemical and biophysical properties of isolated haemosiderins have been compared to that of another iron‐containing protein, termed prehaemosiderin, which sediments through chaotropic potassium iodide only after 20 h of ultracentrifugation, in contrast to that of haemosiderin which is recovered after 2 h of ultracentrifugation. The irodprotein ratio and irodphosphate ratio were less that that of the corresponding haemosiderin, while the elemental composition was also reduced in many of the prehaemosiderin samples. Mossbauer spectroscopy and electron diffraction identified the predominant presence of ferrihydrite in prehaemosiderin species even though the secondary haemochromatosis haemosiderin iron cores were essentially goethite‐like. The majority of the pre‐haemosiderins isolated showed the presence of an additional peptide band at 17 kDa in addition to that at 21 kDa.Further Mossbauer studies of haemosiderin isolated from untreated secondary haemochromatosis patients showed that goethite was the predominant form of iron present, thereby indicating that the presence of this form of ferrihydrite was not wholly attributable to chelation therapy.
7α Hydroxylation of 25‐Hydroxycholesterol in Liver Microsomes
Authors: Toll Anders , Wikvall Kjell , Sudjana‐Sugiaman Elly , Kondo Kazu‐Hiro , Björkhem Ingemar 
Published: March 2005

Rat, pig and human liver microsomes were found to catalyze 7α‐hydroxylation of 25‐hydroxycholesterol. In contrast to cholesterol 7α‐hydroxylase activity, the 7α‐hydroxylase activity towards 25‐hydroxycholesterol in rat liver was not stimulated by cholestyramine treatment. After transfection with cDNA for human cholesterol 7α‐hydroxylase, COS cells showed a significant activity towards cholesterol but not towards 25‐hydroxycholesterol. During purification of cholesterol 7α‐hydroxylase from pig liver microsomes, about 99% of the 7α‐hydroxylase activity towards 25‐hydroxycholesterol and 27‐hydroxycholesterol was clearly separated from 7α‐hydroxylase activity for cholesterol. The small amount of 25‐hydroxycholesterol 7α‐hydroxylase activity retained in a partially purified preparation of cholesterol 7α‐hydroxylase was not inhibited by addition of cholesterol, indicating that the oxysterol binding site is different from the cholesterol binding site, presumely due to the presence of two different enzymes. It is concluded that different enzymes are involved in 7α‐hydroxylation of cholesterol and 7α hydroxylation of side‐chain‐oxidized cholesterol in rat, pig and human liver.Inhibition experiments with a partially purified fraction of the oxysterol 7α‐hydroxylase from pig liver gave results consistent with the contention that the same enzyme is responsible for 7α hydroxylation of both 25‐hydroxycholesterol and 27‐hydroxycholesterol.It has been suggested that cholesterol 7α‐hydroxylase can preferentially use oxysterols, in particular 25‐hydroxycholesterol, as substrates and by this means inactivate important physiological regulators of cholesterol homeostasis. Such a mechanism would explain the unique property of the liver to resist down‐regulation of the low‐density‐lipoprotein receptor [Dueland, S., Trawick, J. D., & Davies, R. A. (1993) J. Biol. Chem. 267, 22695–22698]. The present results do not support the contention that the important coupling between cholesterol 7α‐hydroxylase activity, the low‐density‐lipoprotein receptor activity and hydroxymethylglutaryl coenzyme A reductase activity in liver cells is due to inactivation of 25‐hydroxycholesterol or 27‐hydroxycholesterol by the action of cholesterol 7α‐hydroxylase.
X‐ray Structure of a Pokeweed Antiviral Protein, Coded by a New Genomic Clone, at 0.23 nm Resolution
Authors: Ago Hideo , Kataoka Jiro , Tsuge Hideaki , Habuka Noriyuki , Inagaki Eiji , Noma Masana , Miyano Masashi 
Published: March 2005

We have determined the crystal structure of α‐pokeweed antiviral protein, a member of ribosome‐inactivating proteins, at 0.23 nm resolution, by the molecular‐replacement method. The crystals belong to the space group P21212 with unit‐cell dimensions a= 4.71, b = 11.63 and c= 4.96 nm, and contain one protein molecule/asymmetric unit based on a crystal volume/unit protein molecular mass of 2.1×10−3nm3/Da. The crystallographic residual value was reduced to 17.2% (0.6–0.23 nm resolution) with root‐mean‐square deviations in bond lengths of 1.9 pm and bond angles of 2.2°.The Cα‐Cα distance map shows that α‐pokeweed antiviral protein is composed of three modules, the N‐terminal (Ala1–Leu76), the central (Tyr77–Lys185) and the C‐terminal (Tyr186–Thr266) modules. The substrate‐binding site is formed as a cleft between the central and C‐terminal modules and all the active residues exist on the central module. The electrostatic potential around the substrate‐binding site shows that the central and C‐terminal module sides of this cleft have a negatively and a positively charged region, respectively. This charge distribution in the protein seems to provide a suitable interaction with the substrate rRNA.