bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro
Authors: Lessard Ivan A. D. , Domingo Gonzalo J. , Borges Adolfo , Perham Richard N.
Published: March 2003

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1×106 Da) with the characteristic 532 symmetry of an icosahedral (60‐mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(α2) dimers or 60 heterotetramers (α2β2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit‐binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post‐translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild‐type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95 % and 85 %, respectively. However, only 26 % of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1α and E1β. This represents the first complete post‐translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.
Activation and reaction kinetics of the dimethylamine/coenzyme M methyltransfer in Methanosarcina barkeri strain Fusaro
Authors: Wassenaar Roel W. , Keltjens Jan T. , van der Drift Chris
Published: March 2003

Dimethylamine/5‐hydroxybenzimidazolylcobamide methyltransferase (DMA‐MT) from Methanosarcina barkeri Fusaro catalyzes (Vmax = 4700 nmol min−1 mg−1 protein; kcat = 7.8 s−1) the transfer of a methyl group from dimethylamine (apparent Km = 0.45 mM) to its corrinoid prosthetic group to yield monomethylamine (MMA) and the methylated enzyme. The product, MMA, is a competitive inhibitor of the reaction (apparent Ki = 5.5 mM). The methyl group bound to the corrinoid prosthetic group of DMA‐MT is subsequently transferred to coenzyme M in a reaction mediated by methylcobalamin/coenzyme M methyltransferase isoenzyme II [MT2(II)], which binds with high affinity to DMA‐MT (apparent Km = 0.22 μM). As isolated, DMA‐MT is inactive, but it can enzymically be reactivated by methyltransferase activating protein (MAP), ATP, and hydrogenase. Apart from the established role in corrinoid activation, ATP was found to act as a powerful allosteric effector on the methyltransferase reaction. The results of kinetic studies, supported by the resolution of as‐yet partially purified auxiliary protein fractions, demonstrate that DMA‐MT, MT2(II), MAP, and hydrogenase are the only enzymic components involved in the dimethylamine/coenzyme M methyltransfer in M. barkeri Fusaro.
Oxidative modification of high‐density lipoprotein 3 induced by human polymorphonuclear neutrophils
Authors: Cogny Anne , Paul Jean‐Louis , Surbled Béatrice , Atger Véronique , Lenoble Michèle , Moatti Nicole
Published: March 2003

The function of high‐density lipoproteins (HDLs) in reverse cholesterol transport is impaired if HDLs are subjected to oxidative stress. Polymorphonuclear neutrophils (PMNs), which have been detected in the earliest stages of atherosclerotic lesions, are one of the most likely sources of the reactive oxygen species that cause such stress.

 In this study, we investigated the effect of a PMN oxidative burst on HDL3. We also studied the impact on these events of pentoxifylline, a drug that regulates granulocyte function.

 HDL3 (370 nmol·mL−1 cholesterol‐HDL) was incubated with PMNs (2 × 106·mL−1) in NaCl/Pi in the presence or absence of an iron chelate complex (10 µm Fe‐nitrilotriacetic acid) at 37 °C for 60 min or 24 h. Phorbol myristate acetate (PMA) or formyl‐methionylleucyphenylalanine (fMetLeuPhe) was used to stimulate PMNs. In iron‐free NaCl/Pi medium, PMA‐stimulated PMNs had a 40% lower HDL3 α‐tocopherol content, whatever the incubation time. In NaCl/Pi medium containing iron, there was 80% less HDL3 α‐tocopherol at 60 min, and HDL3 α‐tocopherol had almost disappeared after 24 h. In this latter condition, the amount of thiobarbituric acid‐reactive substances was significantly higher than the respective control HDL3 (P < 0.05) and oxidation of HDL3 by PMA‐stimulated PMNs was associated with cross‐linking of apoprotein AI, which was detected by SDS/PAGE. Similar results were obtained with fMetLeuPhe‐stimulated PMN except that HDL3 α‐tocopherol was consumed much more slowly during the first 60 min. Pretreatment of PMNs with various concentrations of pentoxifylline (0.001–20 mm) led to the concentration‐dependent inhibition of oxidative modification of HDL3 induced by stimulated PMNs. The addition of 20 mm pentoxifylline in the most extreme oxidative stress conditions resulted in 70% of HDL3 α‐tocopherol being maintained, with no formation of thiobarbituric acid‐reactive substances and a lower level of apoprotein AI cross‐linking.

 Thus HDL3 is susceptible to oxidative modifications induced by stimulated PMNs, in the presence of an exogenous source of iron. Pentoxifylline inhibited the oxidative modification of HDL3 by PMNs.
Transcriptional activity and regulatory protein binding of metal‐responsive elements of the human metallothionein‐IIA gene
Authors: Koizumi Shinji , Suzuki Kaoru , Ogra Yasumitsu , Yamada Hirotomo , Otsuka Fuminori
Published: March 2003

Multiple copies of a cis‐acting DNA element, metal‐responsive element (MRE) are required for heavy metal‐induced transcriptional activation of mammalian metallothionein genes. To approach the regulatory mechanism mediated by these multiple elements, we studied the properties of seven MREs located upstream of the human metallothionein‐IIA (hMT‐IIA) gene in detail. Transfection assays of reporter gene constructs each containing one of these MREs as the promoter element revealed that only four MREs can mediate zinc response. With respect to the distribution of active MREs over the promoter region, the hMT‐IIA gene is largely different from the mouse metallothionein‐I gene, suggesting that MRE arrangement is not an important factor for metal regulation. Experiments using various model promoters showed that multiple MRE copies act highly synergistically, supporting the biological significance of the multiplicity. Only the four active MREs efficiently bound the purified transcription factor human MTF‐1, and MRE mutants defective in binding this protein lost the ability to support zinc‐induced reporter gene expression, strongly suggesting that the direct interaction between human MTF‐1 and a set of the selected MREs plays the major role in heavy metal regulation. In protein/DNA binding reactions in vitro, the purified human MTF‐1 was activated by zinc but not by other metallothionein‐inducing heavy metals, supporting the idea that zinc is the direct modulator of human MTF‐1.
The differential specificity of chymotrypsin A and B is determined by amino acid 226
Authors: Hudáky Péter , Kaslik , Gyula , Venekei , István , Gráf László
Published: March 2003

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.
Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015)
Authors: Ferretti Silvia , Grossmann J. Günter , Hasnain S. Samar , Eady Robert R. , Smith Barry E.
Published: March 2003

Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three‐dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of Mr 134 000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X‐ray‐scattering data, analysed using a model‐independent method for shape restoration, which gave a ≈ 20 Å resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was ≈ 6 µmol of N2O reduced·min–1·(mg of protein)–1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven‐line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.
Structural analysis of a novel putative capsular polysaccharide from Pseudomonas (Burkholderia) caryophylli strain 2151
Authors: Molinaro Antonio , De Castro Cristina , Evidente Antonio , Holst Otto , Parrilli Michelangelo
Published: March 2003

A novel putative capsular polysaccharide consisting of d‐Glcp and
d‐Fruf in the molar ratio of 1 : 1 was isolated as minor constituent from the lipopolysaccharide (LPS) fraction of Pseudomonas (Burkholderia) caryophylli. Its structure was determined, using mainly one‐ and two‐dimensional NMR spectroscopy, as:  →6)‐α‐
d‐Glcp‐(1→1)‐β‐
d‐Fruf‐(2→
Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12
Authors: Jaxel Christine , Duguet Michel , Nadal Marc
Published: March 2003

Reverse gyrase is a type I‐5′ topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes place, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA–S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non‐hydrolyzable ATP analog adenosine 5′‐[β,γ‐imido]triphosphate increases it without changing the sequence specificity.
The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1‐independent fashion
Authors: Balcells Lluís , Calero Fernando , Gómez Néstor , Ramos José , Ariño Joaquín
Published: March 2003

We have previously shown that fission yeast encodes a PPZ‐like phosphatase, designated Pzh1, which is an important determinant of cation homeostasis. pzh1Δ mutants display increased tolerance to Na+ ions, but they are hypersensitive to KCl [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ariño, J. (1997) Eur. J. Biochem.250, 476–483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild‐type cells. K+ ion content of pzh1Δ cells is about twice that of wild‐type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high‐affinity transporter of K+ ions. trk1Δ mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na+‐sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.
A novel egg‐derived tyrosine phosphatase, EDTP, that participates in the embryogenesis of Sarcophaga peregrina (flesh fly)
Authors: Yamaguchi Shinji , Homma Ko‐ichi , Natori Shunji
Published: March 2003

We have previously reported that cathepsin L mRNA is present in unfertilized eggs of Sarcophaga peregrina (flesh fly) as a maternal mRNA, which suggests that cathepsin L is required for embryogenesis. Now we have identified an egg protein, with a molecular mass of 100 kDa, that is extremely susceptible to cathepsin L digestion and which disappears rapidly as the embryos develop. We purified this protein to homogeneity, cloned its cDNA, and found that it contained a consensus sequence for the active site of tyrosine phosphatase. In fact this protein showed tyrosine phosphatase activity, indicating that it is a novel tyrosine phosphatase. The expression and subsequent disappearance of this protein, which we have named egg‐derived tyrosine phosphatase (EDTP), may be indispensable for embryogenesis of Sarcophaga.
Purification and characterization of a detergent‐requiring membrane‐bound metalloendopeptidase from porcine brain
Authors: Jeohn Gwang‐Ho , Matsuzaki Hidemi , Takahashi Kenji
Published: March 2003

A detergent‐requiring metalloendopeptidase cleaving a progastrin‐C‐terminal peptide (progastrin‐(88–101)) mainly at the Arg95‐Gly96 bond was solubilized from porcine cerebral vesicular membranes and purified to homogeneity as examined by PAGE. The purified enzyme had a molecular mass of ≈76 kDa as estimated by both SDS/PAGE and Sephacryl S‐300 gel filtration. It hydrolyzed progastrin‐(88–101) peptide, BAM‐12P, and bradykinin fairly specifically, and more efficiently than various other neuropeptides and related oligopeptides examined as substrates. It was inactive in the absence of detergents, and required certain detergents such as Triton X‐100 or Lubrol PX for activity. Its optimum pH was about 6.5 and was strongly inhibited by metal‐chelating agents such as EDTA, EGTA, and o‐phenanthroline. It was extremely sensitive to EDTA and was completely inhibited even by 0.3 µm EDTA; the activity was fully restored by addition of a 10‐fold higher concentration of Zn2+, Co2+, or Mn2+ ions over EDTA. On the other hand, dynorphin A‐(1–13) peptide, a strong inhibitor of neurolysin, failed to inhibit the enzyme. The various characteristics indicated that the present enzyme is a unique membrane‐bound metalloendopeptidase.
Solution structure of the B form of oxidized rat microsomal cytochrome b5 and backbone dynamics via 15N rotating‐frame NMR‐relaxation measurements
Authors: Arnesano Fabio , Banci Lucia , Bertini Ivano , Felli Isabella C. , Koulougliotis Dionysios
Published: March 2003

Cytochrome b5 in solution has two isomers (A and B) differing by a 180° rotation of the protoporphyrin IX plane around the axis defined by the α and γ meso protons. Homonuclear and heteronuclear NMR spectroscopy has been employed in order to solve the solution structure of the minor (B) form of the oxidized state of the protein and to probe its backbone dynamics in the µs–ms timescale in both oxidation states. A family of 40 conformers has been obtained using 1302 meaningful NOEs and 220 pseudocontact shifts and is characterized by high quality and good resolution (rmsd to the mean structure of 0.055 ± 0.009 nm and 0.103 ± 0.011 nm for backbone and heavy atoms, respectively). Extensive comparisons of the structural and dynamics changes associated with the A‐to‐B form interconversion for both oxidation states were subsequently performed. Propionate 6 experiences a redox‐state‐dependent reorientation as does propionate 7 in the A form. Significant insights are obtained into the role of the protein frame for efficient biological function and backbone mobility is proposed to be one of the factors that could control the reduction potential of the heme.
Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II
Authors: Van Dijk Juliette , Furch Marcus , Derancourt Jean , Batra Renu , Knetsch Menno L. W. , Manstein Dietmar J. , Chaussepied Patrick
Published: March 2003

Changes in the actin–myosin interface are thought to play an important role in microfilament‐linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment‐1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G‐actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G‐actin polymerization. The affinity of monomeric actin (G‐actin) was 20‐fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613–623) resulted in equivalent affinities of G‐actin for M765 and for S1. Proteolytic cleavage and cross‐linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F‐actin). For both types of myosin, F‐actin prevents trypsin cleavage in the loop 2 region and F‐actin segment 1–28 can be cross‐linked to loop 2 residues by a carbodiimide‐induced reaction. In contrast with the S1, loop residues 559–565 of D. discoideum myosin was not cross‐linked to F‐actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G‐actin and F‐actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.
The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength
Authors: Goldmann Wolfgang H. , Hess Daniel , Isenberg Gerhard
Published: March 2003

We employed quasi‐elastic light scattering and electron microscopy to investigate the influence of intact talin and talin tail fragment on actin filament dynamics and network structure. Using these methods, we confirm previous reports that intact talin induces cross‐linking as well as filament shortening on actin networks. We now show that the effect of intact talin as well as talin tail fragment on actin networks is controlled by pH and ionic strength. At pH 7.5, actin filament dynamics in the presence of intact talin and talin tail fragment are characterized by a rapid decay of the dynamic structure factor and by a square root power law for the stretched exponential decay which is in contrast with the theory for pure actin solutions. At pH 6 and low ionic strength, intact talin cross‐links actin filaments more tightly than talin tail fragment. Talin head fragment showed no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain.
Intracellular degradation of the HIV‐1 envelope glycoprotein
Authors: Courageot Joël , Fenouillet Emmanuel , Bastiani Paul , Miquelis Raymond
Published: March 2003

Analysis of the fate of HIV‐1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK‐21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120‐kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3‐loop epitope and the C‐terminal domain of gp120 (amino acids 506–516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK‐21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomittant increase in the amount of a 120‐kDa protelolytic fragment. This fragment had the same biochemical characteristics as the 120‐kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Futhermore, there was evidence that changes in redox state are involved in the ER‐dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose‐dependent degradation of the 120‐kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV‐1 envelope glycoprotein may take place in the ER of infected cells.
Properties of a subtilisin‐like proteinase from a psychrotrophic Vibrio species
Authors: Kristjánsson Magnús M. , Magnússon Ólafur Th. , Gudmundsson Haflidi M. , Alfredsson Gudni Á. , Matsuzawa Hiroshi
Published: March 2003

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA‐44) belongs to the proteinase K family of the superfamily of subtilisin‐like proteinases. The enzyme is secreted as a 47‐kDa protein, but under mild heat treatment (30 min at 40 °C) undergoes autoproteolytic cleavage on the carboxyl‐side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47‐kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold‐adapted enzyme against succinyl‐AAPF‐p‐nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2‐oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.
FIRE3 in the promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous transcription factors CBF and USF but does not mediate an insulin response in a rat hepatoma cell line
Authors: Roder Karim , Wolf Siegmund S. , Sickinger Stefan , Schweizer Michael
Published: March 2003

Several putative insulin‐responsive elements (IRE) in the fatty acid synthase (FAS) promoter have been identified and shown to be functional in adipocytes and hepatocytes. Here we report on the insulin‐responsiveness in the rat hepatoma cell line H4IIE of four cis‐elements in the FAS promoter: the FAS insulin‐responsive elements, FIRE2 and FIRE3; the inverted CCAAT element, ICE; and the insulin/glucose‐binding element, designated hepatic FIRE element, hFIRE, originally identified in rat hepatocytes. Using electrophoretic mobility shift assay (EMSA) competition experiments together with supershifts and in vitro transcription/translation we show that FIRE3 (−68/−58) binds not only the upstream stimulatory factors USF‐1/USF‐2 but also the CCAAT‐binding factor CBF, also known as the nuclear factor Y, NF‐Y. The putative IRE FIRE2, which shows sequence similarity to FIRE3, is located between −267 and −249. Gel retardation experiments indicate that USF‐1 and USF‐2 also bind to this element, which contains an imperfect E‐box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBF. Transient transfection experiments using FAS promoter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of these elements mediates the insulin response of the FAS promoter in the rat hepatoma cell line H4IIE, however, ICE at −103/−87 is responsible for mediating the effect of the insulin antagonist cAMP. The hFIRE element located at −57/−34, in spite of its role in the glucose/insulin response in primary rat hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promoters of the FAS genes in rat, human, goose and chicken is conserved regarding CBF‐binding sites and USF‐binding sites implies an important role for these ubiquitously expressed transcription factors in the regulation of the FAS promoter.
Heme and acute inflammation
Authors: Lyoumi Saïd , Puy Hervé , Tamion Fabienne , Bogard Catherine , Leplingard Antony , Scotté Michel , Vranckx Roger , Gauthier Francis , Hiron Martine , Daveau Maryvonne , Nordmann Yves , Deybach Jean Charles , Lebreton Jean Pierre
Published: March 2003

Acute‐phase protein synthesis in the liver during inflammation is regulated via cytokines and glucocorticoids. Using quantitative reverse transcription (RT)–PCR analysis and immunoassay, we explored, in the rat, the response of the acute‐phase protein, alpha‐2 macroglobulin (A2M), after systemic inflammation induced by lipopolysaccharide (LPS) or localized inflammation induced by turpentine oil (TO). The results indicate that synthesis of A2M is higher following TO‐induced inflammation than LPS‐induced inflammation and is not correlated with interleukin (IL)‐6 or glucocorticoid levels. We studied the putative role of heme in this differential A2M expression following localized vs. systemic inflammation; addition of heme during LPS‐induced inflammation can boost the expression of A2M, whereas blocking heme synthesis (by succinyl acetone) or enhancing its consumption in parallel biosynthetic pathways (cytochrome P450 induction by phenobarbital) decreases A2M expression. This decrease was abolished by exogenous heme supplementation. Finally, we demonstrate that heme supplementation is also able to increase the A2M response in female rats to a level similar to that in male rats providing a new insight into the puzzling sexual dimorphism observed previously during localized inflammation. We propose that heme should be considered a new regulatory element in controlling liver A2M expression during inflammation.
Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the α‐subunits of Na+/K+‐ATPase
Authors: Antolovic Roberto , Hamer Evelyn , Serpersu Engin H. , Kost Holger , Linnertz Holger , Kovarik Zeljka , Schoner Wilhelm
Published: March 2003

To test the hypothesis that Na+/K+‐ATPase works as an (αβ)2‐diprotomer with interacting catalytic α‐subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution‐inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C‐terminal Na+‐like 80‐kDa as well as K+‐like 60‐kDa peptides and N‐terminal 40‐kDa peptides of the α‐subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4‐8‐N3‐ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+‐ATPase with an inactivation rate constant (k2) of 12 × 10−3·min−1 at 37 °C and a dissociation constant (Kd) of 207 ± 28 µm. Tryptic digestion of the [γ32P]Co(NH3)4‐8‐N3‐ATP‐inactivated and photolabelled α‐subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+‐like C‐terminal 60‐kDa fragment which was labelled in addition to an unlabelled Na+‐like C‐terminal 80‐kDa fragment. Tryptic digestion of [α32P]‐or [γ32P]Cr(H2O)4ATP – bound to the E1ATP (high affinity) site – led to the labelling of a Na+‐like 80‐kDa fragment besides the immediate formation of an unlabelled K+‐like N‐terminal 40‐kDa fragment and a C‐terminal 60‐kDa fragment. Because a labelled Na+‐like 80‐kDa fragment cannot result from an unlabelled K+‐like 60‐kDa fragment, and because unlabelled α‐subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+‐ and K+‐like conformations are stabilized by tight binding of substitution‐inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (αβ)2‐diprotomeric Na+/K+‐ATPase.
Identification of novel sites in the ovine growth hormone receptor involved in binding hormone and conferring species specificity
Authors: Allan Gordon J. , Shand John H. , Beattie James , Flint David J.
Published: March 2003

Using site‐directed mutagenesis we mutated the extracellular domain of the ovine growth hormone receptor (oGHR) to the corresponding amino acids in the rat GHR at two different sites: site A is between Thr28 and Leu34 and represents a major immunogenic epitope, while site B is between Ser121 and Asp124 and is involved in the interaction of the human GHR with growth hormone (GH). Native and mutant receptors were bacterially expressed and refolded, and then RIA and GH‐binding assays were carried out on the purified recombinant proteins. Mutations at the N‐terminal site A of oGHR led to greatly reduced binding to bovine GH and, in addition, to significant loss of recognition by a polyclonal antiserum to bovine GHR which recognizes site A as a major epitope. The crystal structure of human GH bound to human GHR did not resolve this extreme N‐terminal region of the receptor but our data indicate that the N‐terminal loop undertakes a 180° turn bringing it into close proximity to the hormone‐binding domain in a fashion analogous to the prolactin receptor. A fourfold decrease in affinity for binding bovine GH was also observed after mutation of site B. However, this change from the ovine sequence to the equivalent sequence in the rat GHR at site B caused a 2.4‐fold increase in the affinity of binding to rat GH. Taken together, the changes in binding affinity of the site‐B mutant for rat and bovine GH demonstrate that this site is involved in conferring species specificity for binding GH.
NFκB activation is required for interferon regulatory factor‐1‐mediated interferon β induction
Authors: Kirchhoff Sabine , Wilhelm Dagmar , Angel Peter , Hauser Hansjörg
Published: March 2003

The interferon regulatory factor 1 (IRF‐1) acts as a transcriptional inducer of the interferon β (IFN‐β) gene and interferon‐stimulated genes. Here we report that IRF‐1‐mediated IFN‐β induction depends on NFκB activity. IRF‐1 by itself initiates NFκB activation by inducing a reduction in cellular MAD3/IκBα, an inhibitor of NFκB. After nuclear translocation, NFκB synergizes with IRF‐1 on the cis‐elements positive regulatory domain (PRD)II and PRDI/III to induce transcription of the IFN‐β gene. In contrast with IFN‐β transcription induced by dsRNA or virus, c‐Jun/ATF‐2 binding to PRDIV is not involved. Recombinant MAD3/IκBα is phosphorylated in vitro by extracts from IRF‐1‐expressing cells. IRF‐1‐dependent MAD3/IκBα degradation is not detectable in cells expressing a dominant negative mutant of the protein kinase PKR, suggesting that PKR mediates MAD3/IκBα degradation.
The effect of Glu75 of staphylococcal nuclease on enzyme activity, protein stability and protein unfolding
Authors: Chen Hueih Min , Dimagno Theodore J. , Wang Wei , Leung Eric , Lee Cheng‐Hao , Chan Sunney I.
Published: March 2003

Staphylococcal nuclease mutants, E57G and E75G, were generated. A comparison of the kinetic parameters both for mutants and wild‐type protein shows that the Michaelis constants (Km) were almost identical for the wild‐type protein and E57G mutant. An approximately 30‐fold decrease in Km compared with the wild‐type protein was observed for the E75G mutant. The turnover numbers for the enzyme (kcat) were higher with both the wild‐type protein and the E57G mutant (3.88 ± 0.21 × 103 s−1 and 3.71 ± 0.28 × 103 s−1) than with the E75G mutant (3.04 ± 0.02 × 102 s−1). The results of thermal denaturation with differential scanning microcalorimetry indicate that the excess calorimetric enthalpy of denaturations, ΔHcal, was almost identical for the wild‐type protein and E57G mutant (84.1 ± 6.2 kcal·mol−1 and 79.3 ± 7.1 kcal·mol−1, respectively). An approximately twofold decrease in ΔHcal compared with the wild‐type protein was observed for the E75G mutant (42.7 ± 5.5 kcal·mol−1). These outcomes imply that Glu at position 75 plays a significant role in maintaining enzyme activity and protein stability. Further study of the unfolding of the wild‐type protein and E75G mutant was conducted by using time‐resolved fluorescence with a picosecond laser pulse. Two fluorescent lifetimes were found in the subnanosecond time range. The faster lifetime (τ2) did not generally vary with either pH or the concentration of guanidinium hydrochloride (GdmHCl) in the wild‐type protein and the E75G mutant. The slow lifetime (τ1), however, did vary with these parameters and was faster as the protein is unfolded by either pH or GdmHCl denaturation. The midpoints of the transition for τ1 are pH 3.5 and 5.8 for the wild‐type protein and E75G mutant, respectively, and the GdmHCl concentrations are 1.1 m and 0.6 m for the wild‐type protein and E75G mutant, respectively. Parallel steady‐state fluorescence measurements have also been carried out and the results are in general agreement with the time‐resolved fluorescence experiments, indicating that Glu at position 75 plays an important role in protein unfolding.
Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3
Authors: Hanck Theodor , Stricker Rolf , Krishna U. Murali , Falck John R. , Chang Young‐Tae , Chung Sung‐Kee , Reiser Georg
Published: March 2003

We have recently cloned the cDNA of p42IP4, a membrane‐associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hülser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett.405, 229–236.] p42IP4 is a protein of 374 amino acids with Mr of 42 kDa. The p42IP4 protein has a zinc finger motif at its N‐terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42IP4, it was expressed as a glutathione‐S‐transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione‐S‐transferase with the protease factor‐Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42IP4, purified from pig cerebellum or rat brain (Ki for inositol(1,3,4,5)P4 of 4.1 nm and 2.2 nm, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p42IP4 binds phosphatidylinositol(3,4,5)P3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P3; this binding occurs in a concentration‐dependent manner and is blocked by inositol(1,3,4,5)P4. This specific association and the possibility that endogenous p42IP4 can be converted from a membrane‐associated state to a soluble state support the hypothesis that p42IP4 might be redistributed between cellular compartments upon hormonal stimulation.
Contribution of a prosegment lysine residue to the function and structure of porcine pepsinogen A and its active form pepsin A
Authors: Richter Catherine , Tanaka Takuji , Koseki Taihei , Yada Rickey Y.
Published: March 2003

A conserved lysine residue, Lys36p, on the prosegment of pepsinogen was replaced with a positively charged arginine (K36pR), a negatively charged glutamic acid (K36pE), and a neutral side chain methionine (K36pM). K36pM and K36pE mutants were extremely unstable and degraded rapidly, especially K36pE, which was inactivated during purification. This instability was confirmed by microcalorimetry where the denaturing temperatures for K36pM and K36pE were 6 °C and 10 °C lower than the wild‐type, respectively. As a function of pH, K36pM and K36pR were activated over a broader range of pH as compared with wild‐type. The mutant pepsinogens were activated faster than wild‐type with K36pM being activated ≈10 times faster. The activated pepsins from the various mutant pepsinogens showed lower kinetic efficiency than wild‐type enzyme. Catalytic rate constants were reduced by half. The results suggested Lys36p is important for the correct folding of the active‐centre residues. The molecular modeling calculation suggested that the position of Asp215 was substantially altered. In conclusion, the above results would suggest that Lys36p was important not only for stability of the prosegment and pepsinogen, but also for the correct alignment of the active‐centre residues.
Identification of subunit g of yeast mitochondrial F1F0‐ATP synthase, a protein required for maximal activity of cytochrome c oxidase
Authors: Boyle Glen M. , Roucou Xavier , Nagley Phillip , Devenish Rodney J. , Prescott Mark
Published: March 2003

By means of a yeast genome database search, we have identified an open reading frame located on chromosome XVI of Saccharomyces cerevisiae that encodes a protein with 53% amino acid similarity to the 11.3‐kDa subunit g of bovine mitochondrial F1F0‐ATP synthase. We have designated this ORF ATP20, and its product subunit g. A null mutant strain, constructed by insertion of the HIS3 gene into the coding region of ATP20, retained oxidative phosphorylation function. Assembly of F1F0‐ATP synthase in the atp20‐null strain was not affected in the absence of subunit g and levels of oligomycin‐sensitive ATP hydrolase activity in mitochondria were normal. Immunoprecipitation of F1F0‐ATP synthase from mitochondrial lysates prepared from atp20‐null cells expressing a variant of subunit g with a hexahistidine motif indicated that this polypeptide was associated with other well‐characterized subunits of the yeast complex. Whilst mitochondria isolated from the atp20‐null strain had the same oxidative phosphorylation efficiency (ATP : O) as that of the control strain, the atp20‐null strain displayed approximately a 30% reduction in both respiratory capacity and ATP synthetic rate. The absence of subunit g also reduced the activity of cytochrome c oxidase, and altered the kinetic control of this complex as demonstrated by experiments titrating ATP synthetic activity with cyanide. These results indicate that subunit g is associated with F1F0‐ATP synthase and is required for maximal levels of respiration, ATP synthesis and cytochrome c oxidase activity in yeast.
Purification of receptor complexes of interleukin‐10
Authors: Hoover David M. , Schalk‐Hihi Céline , Chou Chuan‐Chu , Menon Satish , Wlodawer Alexander , Zdanov Alexander
Published: March 2003

Interleukin‐10 (IL‐10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL‐10 (vIL‐10) is a homologue of human IL‐10 (hIL‐10) produced by Epstein‐Barr virus (EBV). Both hIL‐10 and vIL‐10 bind to the soluble extracellular fragment of the cytokine receptor IL‐10R1 (shIL‐10R1). The stoichiometry of the vIL‐10 : shIL‐10R1 complex has been found to be the same as hIL‐10 : shIL‐10R1, with two vIL‐10 dimers binding to four shIL‐10R1 monomers. Complexes of both hIL‐10 and vIL‐10 with glycosylated shIL‐10R1 could not be crystallized. Controlled deglycosylation using peptide : N‐glycosidase F and endo‐β‐N‐acetylglucosaminidase F3 resulted in the formation of crystals of both hIL‐10 : shIL‐10R1 and vIL‐10 : shIL‐10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand‐receptor complexes should facilitate our understanding of the basis of the interaction between IL‐10 and the IL‐10 receptor.
Characterization of a chromatin remodelling activity in Xenopus oocytes
Authors: Gelius Birgitta , Wade Paul , Wolffe Alan , Wrange Örjan , Östlund Farrants Ann‐Kristin
Published: March 2003

The yeast SWI2/SNF2 protein is a component of a large protein complex which is involved in the remodelling of chromatin during transcriptional activation. Several homologous complexes have been found in Drosophila and mammals. We have examined the expression of the SWI2/SNF2 homologue BRG1 in Xenopus laevis using two antisera originally raised against the C‐terminus of the rat and the human BRG1 protein. These two antisera cross‐reacted with a protein found in both Xenopus liver and Xenopus oocytes. The Xenopus BRG1‐like protein is expressed throughout oogenesis (stages I–VI) and embryogenesis. By injecting an expression vector containing the full‐length human BRG1 cDNA into Xenopus oocytes, the relative molecular weight (Mr) of the Xenopus BRG1‐like protein was shown to be slightly lower than that of the human BRG1, 190 000 and 200 000, respectively. The Xenopus BRG1‐like protein elutes at a Mr of ≈ 2 000 000 on Superose HR6™ size‐exclusion chromatography, indicating that it is part of a larger complex, as are all other known SWI/SNF proteins. Nucleosome remodelling activity was co‐eluted with the BRG1 immunogenic activity in both ion‐exchange and size‐exclusion chromatography.
Glyceraldehyde‐3‐phosphate dehydrogenase from Ehrlich ascites carcinoma cells
Authors: Bagui Swapna , Ray Manju , Ray Subhankar
Published: March 2003

Glyceraldehyde‐3‐phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5–9.0 with an optimum pH of 8.4–8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde‐3‐phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde‐3‐phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells.
Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction
Authors: Suzuki Toshikazu , Iwazaki Ayano , Katagiri Hideki , Oka Yoshitomo , Redpath J. Leslie , Stanbridge Eric J. , Kitagawa Takayuki
Published: March 2003

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three γ‐ray‐induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2‐deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N‐glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2‐deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.
A thermodynamic study of the 434‐repressor N‐terminal domain and of its covalently linked dimers
Authors: Ruiz‐Sanz Javier , Simoncsits András , Törö Imre , Pongor Sandor , Mateo Pedro L. , Filimonov Vladimir V.
Published: March 2003

The isolated N‐terminal 1–69 domain of the 434‐phage repressor, R69, and its covalently linked (head‐to‐tail and tail‐to‐tail) dimers have been studied by differential scanning microcalorimetry (DSC) and CD. At neutral solvent conditions the R69 domain maintains its native structure, both in isolated form and within the dimers. The stability of the domain depends highly upon pH within the acidic range, thus at pH 2 and low ionic strength R69 is already partially unfolded at room temperature. The thermodynamic parameters of unfolding calculated from the DSC data are typical for small globular proteins. At neutral pH and moderate ionic strength, the domains of the dimers behave as two independent units with unfolding parameters similar to those of the isolated domain, which means that linking two R69 domains, either by a long peptide linker or by a designed C‐terminal disulfide bridge, does not induce any cooperation between them.