bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of elongation factor‐2 by multisite phosphorylation
Authors: REDPATH Nicholas T. , PRICE Nigel T. , SEVERINOV Konstantin V. , PROUD Christopher G. 
Published: March 2005

We have studied the phosphorylation of protein synthesis elongation factor eEF‐2, the effects of phosphorylation on its activity and dephosphorylation of phosphorylated eEF‐2 by protein phosphatases‐2A and ‐2C. Extensive analysis of phosphopeptides generated from eEF‐2 phosphorylated in vitro, by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates: in this system mono‐phosphorylated eEF‐2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono‐phosphorylated eEF‐2 detected are phosphorylated at Thr56.The eEF‐2 kinase could phosphorylate a synthetic peptide based on residues 49–60 of eEF‐2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high Km, compared to eEF‐2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53.In the light of the existence of two phosphorylation sites in eEF‐2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)‐directed synthesis of polyphenylalanine, where both the bis‐ and mono‐phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non‐phosphorylated eEF‐2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF‐2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained.Protein phosphatases‐2A and ‐2C (PP‐2A and PP‐2C) can each efficiently dephosphorylate phosphorylated eEF‐2. While bis‐phosphorylated eEF‐2 was a better substrate for PP‐2A than mono‐phosphorylated factor (phosphorlated at Thr56), the converse was true for PP‐2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP‐2C by the presence of phosphate on Thr58. Nevertheless, PP‐2C exhibited a preference for dephosphorylation of Thr56 in bis‐phosphorylated eEF‐2, while PP‐2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.
The rat mitochondrial 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A‐synthase gene contains elements that mediate its multihormonal regulation and tissue specificity
Authors: GIL‐GÓMEZ Gabriel , AYTÉ José , HEGARDT Fausto G. 
Published: March 2005

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A (HMG‐CoA) synthase, a liver‐specific enzyme, is a constituent of the HMG‐CoA cycle responsible for ketone‐body synthesis.We report the isolation and characterization of genomic clones that encompass the gene for rat mitochondrial HMG‐CoA synthase. The gene spans at least 24 kbp and contains ten exons and nine introns. The 5′ flanking region of the gene has also been cloned and characterized. Exon 1 contains the untranslated sequence of the transcript, extending downstream to enclose the coding region for the putative mitochondrial‐targeting signal (35 amino acids). The 1149‐bp proximal region of the transcription start point permits transcription of a reporter gene in transfected hepatoma cells but not in an extrahepatic cell line, confirming the function of the promoter. A truncated construct of 142 bp is still able to promote transcription in hepatoma cells, suggesting the presence of liver‐specific enhancer elements in the proximal promoter region. The 5′ flanking region contains typical promoter elements, including a TATA box and several putative recognition sequences for transcription factors involved in controlling both basal‐level and hormone‐modulated transcription rates. Furthermore, the presence in the mitochondrial HMG‐CoA‐synthase promoter of cis‐elements, responsible for the multihormonal regulation of transcription, is supported by transient transfection experiments.
Isolation and characterization of a cDNA clone encoding a Pleurodeles lectin
Authors: TIFFOCHE Christophe , CHESNEL Amand , JEGO Patrick , LE PENNEC Jean‐Paul 
Published: March 2005

A cDNA encoding a lectin secreted by the oviduct of Pleurodeles Waltl has been isolated and sequenced. The cDNA was identified by comparing the N‐terminal amino‐acid sequence of the purified P. Waltl lectin polypeptides with the amino‐acid sequence deduced from the cDNA. The two chains of the mature protein can be encoded within a unique mRNA. Two mRNA were also found in the oviduct extracts. However, they probably result from differential polyadenylation events. The mRNA are strictly localized in the anterior part of the oviduct and increase after estradiol stimulation, two characteristics which have been previously demonstrated for the protein. P. Waltl is known to possess a very high DNA content (∼2×1010bp) but the aforementioned results and Southern‐blot experiments suggest a unique or a least a very low gene‐copy number for this protein.The amino‐acid sequence of the P. Waltl lectin deduced from the cDNA sequence shows similarities with the C‐type carbohydrate‐recognition domains of animal lectins as defined by Drickamer [Drickamer, K. (1988) J. Biol. Chem. 263, 9557–9560]. Although it is regulated by estradiol, the P. Waltl lectin amino‐acid sequence shows a higher similarity with animal lectins involved in the defence of the organism than with those involved in reproduction and development.
cDNA cloning and mitochondrial import of the β‐subunit of the human electron‐transfer flavoprotein
Authors: FINOCCHIARO Gaetano , COLOMBO Irma , GARAVAGLIA Barbara , GELLERA Cinzia , VALDAMERI Giulio , GARBUGLIO Nicoletta , DIDONATO Stefano 
Published: March 2005

We have isolated a cDNA clone which encodes the entire β‐subunit of human electron‐transferring flavoprotein (ETF) by screening an expression library from human liver using polyclonal antibodies against porcine ETF. This cDNA encodes a protein of 255 amino‐acid residues with a predicted molecular mass of 27 877 Da which shows a high degree of similarity with partial amino acid sequences obtained from both rat liver and Paracoccus denitrificansβ‐ETF. Northern‐blot analysis shows that the human β‐ETF mRNA is approximately 1 kb in size and is abundant in liver, heart and skeletal muscle.Incubation with intact mitochondria indicates that the cDNA‐encoded β‐ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that β‐ETF, unlike the majority of nuclear‐encoded mitochondrial matrix proteins, does not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus demonstrating that it is an energy‐dependent process.Interestingly, the sequence analysis of β‐ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen‐fixing bacterium Azorhizobium caulinodans.
Inhibitor‐2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase‐1 into a conformation with the specificity and regulatory properties of the native enzyme
Authors: ALESSI Dario R. , STREET Alasdair J. , COHEN Philip , COHEN Patricia T. W. 
Published: March 2005

Three isoforms of mammalian protein phosphatase‐1 (PP1α, PP1β and PP1γ) were expressed in Escherichia coli and purified to near homogeneity. The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor‐2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues. Like PP1C, they each formed a complex with the glycogen‐targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1C. The expressed isoforms were 100–600‐fold less sensitive to inhibitor‐1, 3–5‐fold less sensitive to okadaic acid, 5–100‐fold less sensitive to microcystin‐LR and approximately 20‐fold more active in dephosphorylating histone H1 than native PP1C. Although PP1γ (like PP1C) was active in the absence of Mn2+, expressed PP1α and PP1β were completely dependent on Mn2+ for activity. PP1β, like PP1C, interacted with the myofibrillar‐targetting(M) complexes from skeletal‐muscle and smooth‐muscle producing species with enhanced myosin‐phosphatase activity, whereas expressed PP1α and PP1γ did not.The expressed isoforms of PP1 combined with inhibitor‐2 to form an inactive complex (PP1I) that could be reactivated by the glycogen‐synthase‐kinase‐3(GSK3)‐catalysed phosphorylation of inhibitor‐2. This procedure transformed the properties of all three expressed isoforms to those of native PP1C. Their sensitivities to inhibitor‐1, okadaic acid and microcystin‐LR were increased greatly, their histone‐phosphatase activities decreased and the activities of PP1α and PP1β became independent of Mn2+. Furthermore PP1α and PP1γ now interacted with the M complexes in a similar manner to PP1β and PP1C. Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn2+‐dependent form with properties similar to those of the expressed isozymes.The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G‐subunit/PP1C and M‐complex/PP1C heterodimeric complexes. Inhibitor‐2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+. Taken together, the results suggest that inhibitor‐2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.
Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase
Authors: STEFFENS Guy C. M. , SOULIMANE Tewfik , WOLFF Gerhard , BUSE Gerhard 
Published: March 2005

The early observation of extra copper in preparations of cytochrome‐c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations (heme A contents close to the theoretical value of 9.79 nmol/mg protein for the 13‐subunit bovine enzyme) protein‐related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with Mr 204266 were determined.Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one‐electron‐accepting/donating species in the ligand‐free enzyme.Participation of two copper ions in a binuclear copper site acting as a one‐electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the C‐terminal sequence of subunit II with one of the copper‐binding sites in nitrous‐oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.
Mass determination of 15‐hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)‐12‐hydroxy‐5,8,10‐heptadecatrienoic acid as substrate
Authors: HÖHL Wolfgang , STAHL Bernd , MUNDKOWSKI Ralf , HOFMANN Ute , MEESE Claus O. , KUHLMANN Ulrich , SCHLEGEL Werner 
Published: March 2005

NAD+‐dependent 15‐hydroxyprostaglandin dehydrogenase catalyzes the first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have examined enzyme kinetics of the purified enzyme with (5Z,8E,10E,12S)‐12‐hydroxy‐5,8,10‐ heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtration on FPLC. We could estimate a molecular mass of 28 ± 1 kDa, indicating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix‐assisted laser desorption/ionization mass spectrometry to 28740 ± 30 Da.(5Z,8E,10E)‐12‐oxo‐5,8,10‐heptadecatrienoic acid (oxo‐HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 μM, Vi= 12.78 mU/μg] were in agreement with those determined for NADH formation (Km= 7.65 μM, Vi= 11.79 mU/μg). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 (PGE2). As the product oxo‐HT is a potent inhibitor of platelet aggregation, dehydrogenation of HHT might represent a biological activation step.
Interactions between glucose metabolism and oxidative phosphorylations on respiratory‐competent Saccharomyces cerevisiae cells
Authors: BEAUVOIT Bertrand , RIGOULET Michel , BUNOUST Odile , RAFFARD Gérard , CANIONI Paul , GUÉRIN Bernard 
Published: March 2005

The purpose of this work was to analyze the interactions between oxidative phosphorylations and glucose metabolism on yeast cells aerobically grown on lactate as carbon source and incubated in a resting cell medium. On such respiratory‐competent yeast cells, four different metabolic steady states have particularly been studied: (a) glucose feeding under anaerobiosis, (b) ethanol supply under aerobiosis, (c) glucose supply under aerobiosis and (d) glucose plus ethanol under aerobiosis. For each condition, we measured: (a) the cellular ATP/ADP ratio and NADH content sustained under these conditions, (b) the glucose consumption rate (glucose conditions) and the respiratory rate (aerobic conditions).Under aerobic conditions, when ethanol is used as substrate, the ATP/ADP ratio and NADH level are very high as compared with glucose feeding. However, the rate of oxygen consumption is similar under both conditions. The main observation is a large increase in the respiratory rate when both glucose and ethanol are added. This increase corresponds to an ATP/ADP ratio and a NADH level lower than those observed with ethanol but higher than those with glucose. Therefore the response of the respiratory rate to the ATP/ADP ratio depends on the redox potential. We studied the way in which the ATP‐consuming activity was increased under glucose + ethanol conditions. By NMR experiments, it appears that neither the futile cycle at the level of the phosphofructo‐1‐kinase/fructo‐1,6‐bisphosphatase couple nor the synthesis of carbohydrate stores could account for the increase in oxidative phosphorylation. However, it is shown that, in the presence of glucose + ethanol, ATP consumption is strongly stimulated. It is hypothesized that this consumption is essentially due to the combination of the well‐known plasma membrane proton‐ATPase activation by glucose and the high phosphate potential due to oxidative ethanol metabolism. While it is well documented that oxidative phosphorylations inhibit the glycolytic flux, i.e. the Pasteur effect, we clearly show in this work that the glycolytic pathway limits the ability of mitochondria to maintain a cellular phosphate potential.
Molecular cloning, sequence analysis and expression of the gene for catalase‐peroxidase (cpeA) from the photosynthetic bacterium Rhodobacter capsulatus B10
Authors: FORKL Hubert , VANDEKERCKHOVE Joel , DREWS Gerhart , TADROS Monier Habib 
Published: March 2005

The gene encoding catalase‐peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10. The nucleotide sequence of a 3.7‐kb SacI–HindIII fragment, containing the catalase‐peroxidase gene (cpeA) and its flanking regions were determined. A 1728‐bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed. A Shine‐Dalgarno sequence was found 5 bp upstream from the translational start site. The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase‐peroxidase of R. capsulatus B10.The amino acid sequence of R. capsulatus catalase‐peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase‐peroxidase of Mycobacterium intracellulare (42.2%). As shown by a cpeA::lacZ fusion in trans in R. capsulatus, the expression of the catalase‐peroxidase gene is regulated by oxygen. The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.
Zn‐exchange and Mössbauer studies on the [Fe‐Fe] derivatives of the purple acid Fe(III)‐Zn(II)‐phosphatase from kidney beans
Authors: SUERBAUM Hildegard , KÖRNER Michael , WITZEL Herbert , ALTHAUS Erika , MOSEL Bernd‐Dieter , MÜLLER‐WARMUTH Werner 
Published: March 2005

In order to perform Mössbauer studies, Zn(II) in the Fe(III)‐Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)‐57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)‐57Fe(III) species with a 30‐times lower activity. Incubation of the metal‐free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)‐57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)‐57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)‐Fe(II)/Fe(III)‐Fe(III) species, but not the mixed valent Fe(III)‐Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III).The Mössbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with δ of 0.51 mm/s and 0.53 mm/s and E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a μ‐oxo bridge. The mixed‐valent Fe(III)‐Fe(II) species shows two quadrupole doublet with δ values of 0.55 mm/s and 1.14 mm/s and E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half‐integer spin system with antiferromagnetic coupling. The Fe(II)‐Fe(II) system exhibits two quadrupole doublets with δ values of 1.18 mm/s and 1.22 mm/s and with E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)‐binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)‐Fe(II) system. Interaction with O2 is discussed.
Characterization of a transcriptional control element involved in proliferation of peroxisomes in yeast in response to oleate
Authors: EINERHAND Alexandra W. C. , KOS Wilko T. , DISTEL Ben , TABAK Henk F. 
Published: March 2005

Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the proliferation of these organelles in Saccharomyces cerevisiae. Previously, we have reported the identification of a region containing a positive regulatory element in the 5′ flanking region of the FOX3 gene encoding the peroxisomal enzyme 3‐oxoacyl‐CoA thiolase. This region contains a 23‐bp imperfect inverted‐repeat sequence. Full induction, in response to oleate, is mediated by the intact dyad. However, one half‐site of the inverted repeat is also able to mediate induction of transcription in response to oleate, albeit to a small extent. Furthermore, the weak binding of protein to each part of the inverted repeat proved to be correlated with the weak activation of transcription, in support of oleate.A DNase‐I footprint covered the entire dyad and DNA band‐shift experiments indicated that one or more trans‐acting factors bind to the imperfect palindrome. The binding of protein to this element seems to be correlated with transcriptional activation, since mutations in both halves of the inverted dyad affected both transcriptional activation and protein binding in vitro.Similar oleate‐responsive elements are commonly found in the 5′ flanking regions of genes encoding proteins involved in peroxisome biogenesis and the factor(s) binding to oleate‐responsive element(s) could therefore be involved in coordination of the expression of oleate‐inducible genes and the proliferation of peroxisomes.
Mammalian PC‐12 cell genetically engineered for human cytochrome P450 2E1 expression
Authors: MAPOLES John , BERTHOU François , ALEXANDER Alice , SIMON Francis , MÉNEZ Jean‐François 
Published: March 2005

The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC‐12. This cell line expressed cytochrome b5 (58 ± 12 pmol/mg microsomal protein vs 528 ± 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 ± 20 nmol · min−1· mg microsomal protein−1 vs 68 ± 48 nmol · min−1· mg−1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co‐transfected with the plasmid RSVneo into PC‐12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6‐hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB‐7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC‐12 cells. Chlorzoxazone 6‐hydroxylation, n‐butanol oxidation and dimethylnitrosamine N‐demethylation were localized in microsomes (62, 60 and 63 pmol · min−1· mg microsomal protein−1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB‐7 cells has catalytic competence similar to human liver CYP2E1. DB‐7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase.Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.