bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation Effects on Flanking Charged Residues
Authors: Chavanieu Alain , Keane Noeleen E. , Quirk Philip G. , Levine Barry A. , Calas Bernard , Wei Lei , Ellis Leland
Published: March 2005

1H‐NMR and 31P‐NMR spectroscopy were employed to assess the electrostatic consequences of phosphorylation of single and multiple tyrosine residues in peptides derived from the core and tail autophosphorylation regions of the human insulin receptor tyrosine‐kinase domain. In both peptides, phosphorylation was accompanied by changes in the resonances from basic side‐chains; those from acidic residues were unaffected. Tyrosine phosphorylation caused increases of up to one in the pKa values of histidine residues situated up to eight residues away in the primary sequence. Titration curve analysis by Hill plots suggested some cooperativity of histidine and phosphate ionizations. Behaviour closely analogous to that of the insulin receptor tail peptide was observed during changes in phosphorylation of the intact insulin receptor kinase domain, suggesting that the electrostatic dissemination effects seen for the isolated peptide are retained by the peptide sequence in the context of the much larger protein. Similar changes in the behaviour of basic residues were also observed upon tyrosine phosphorylation of a cdc2‐derived peptide, suggesting that this potential of phosphorylation events to propagate directed structural changes may find a widespread utility in the activation of protein kinases and in the transduction of phosphorylation‐based signalling.
Characterization of the Interaction Between Plasminogen and Staphylokinase
Authors: Lijnen H. Roger , De Cock Frans , Van Hoef Berthe , Schlott Bernhard , Collen Désiré
Published: March 2005

Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka=ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real‐time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu‐plasminogen was 0.93×108M‐1 as compared to 2.0×108M‐1 and 1.6×108M‐1, respectively, for the binding to [S741A]recombinant plasminogen or Lys‐[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active‐site blocked plasmin occurred with Ka, values of 4.0×108M‐1 and 8.4×108M‐‐1, respectively, whereas active‐site blocked LMM‐plasmin (a plasmin derivative lacking kringles 1– 4) and the plasmin B‐chain bound with Ka values of 1.0×108M‐1 and 0.49×108M‐1, respectively. Lysine‐binding site I (a plasminogen derivative consisting of kringles 1–3) and lysine‐binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2×105M‐1 and 2.9×105M‐1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin‐SakSTAR complex to streptokinase and binding of the plasmin‐streptokinase complex to SakSTAR, were, respectively, 44‐fold and 30‐fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site‐specific mutagenesis of Met26 to arginine or alanine) were 10–20‐fold lower than that of native staphylokinase.

These results indicate that: (a) the affinity of staphylokinase for Glu‐plasminogen and Lysplasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1– 4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high‐affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.
Flavodoxin is Required for Conversion of Dethiobiotin to Biotin in Escherichia coli
Authors: Ifuku Ohji , Koga Nobuyoshi , Haze Shin‐ichiro , Kishimoto Jiro , Wachi Youji
Published: March 2005

We have reported [Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M. & Fukushima, S. (1992) Biosci. Biotechnol. Biochem. 56, 1780–1785] the enzymic conversion of dethiobiotin to biotin (catalyzed by the enzyme encoded by bioB) in cell‐free extract of Escherichia coli which had been genetically engineered for high bioB expression. An unidentified protein(s) in addition to the bioB gene product is obligatory for this reaction. We have found that this protein was precipitated from the cell‐free extract with poly(ethyleneimine), and we have purified it to homogeneity by a procedure which includes ammonium sulfate fractionation, DEAE‐cellulose chromatography, gel filtration, and Mono Q chromatography. The apparent molecular mass of the purified protein was estimated to be about 21 kDa by SDS/PAGE. The N‐terminal amino acid sequence of the purified protein was identical with that of E. coli flavodoxin. We conclude that flavodoxin is required for conversion of dethiobiotin to biotin in E. coli. Studies with purified flavodoxin and the fraction containing the bioB gene product suggested that protein(s) in addition to the bioB gene product and flavodoxin is also obligatory for the reaction.
Isolation, Characterization and Structural Organization of the Gene and Pseudogene for the Dihydrolipoamide Succinyltransferase Component of the Human 2‐Oxoglutarate Dehydrogenase Complex
Authors: Nakano Kyoko , Takase Chiyoshi , Sakamoto Takashi , Nakagawa Shiro , Inazawa Johji , Ohta Shigeo , Matuda Sadayuki
Published: March 2005

In the present study, the dihydrolipoamide succinyltransferase gene of the 2‐oxoglutarate dehydrogenase complex was isolated from a human genomic DNA library and its entire nucleotide sequence was determined. This gene was approximately 23 kbp in size with 15 exons and 14 introns. All of the donor and acceptor splice sites of this gene conformed to the GT/AG rule. A quanine residue 43 bases upstreams of the ATG initiating translation codon was the transcription initiation site of the human dihydrolipoamide succinyltransferase mRNA. Sequence analysis of the promoter‐regulatory region showed the presence of a CAAT‐box‐like sequence but the presence of a TATA‐box‐like sequence was not evidenced. Also located in this region were sequences resembling glucocorticoid‐responsive and CAMP‐responsive elements, and an Sp1 binding site. No nucleotide sequence corresponding to the E3‐binding and/or E1‐binding domain was found in any region of the gene. Therefore, the exon coding for the E3‐binding and/or E1‐binding domain may have been lost from the gene during evolution. Moreover, a processed pseudogene of dihydrolipoamide succinyltransferase was isolated and sequenced. The nucleotide sequence of the pseudogene is 93% similar to the sequence of the human dihydrolipoamide succinyltransferase cDNA, but the pseudogene is not functional for base changes, deletions and insertions of the pseudogene. Southern‐blot analysis showed the presence of a single copy of this gene and a single copy of a pseudogene in the human genome. In addition, a possible relationship between dihydrolipoamide succinyltransferase and familial Alzheimer's disease is discussed.
Conversion of Enkephalin and Dermorphin into δ‐Selective Opioid Antagonists by Single‐Residue Substitution
Authors: Tancredi Teodorico , Salvadori Severo , Amodeo Pietro , Picone Delia , Lazarus Lawrence H. , Bryant Sharon D. , Guerrini Remo , Marzola Giuliano , Temussi Piero A.
Published: March 2005

The properties of di‐ and tri‐peptides containing 1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid (Tic) in second position suggest that the message domain of opioid peptides can be composed of only two residues [Temussi, P. A., Salvadori, S., Amodeo, P., Guerrini, R., Tomatis, R., Lazarus, L. H., Picone, D. & Tancredi, T. (1994) Biochem. Biophys. Res. Commun. 198, 933–9391. As a crucial test of the possibility that the Tyr‐Tic segment be a message domain in longer peptide sequences, we have inserted it in the sequences of two typical opioid peptides: [Leu]enkephalin, a non‐selective agonist, and dermorphin, a selective μ agonist. Here we report the synthesis and biological activity of [l‐Tic2]enkephalin, [l‐Tic2]dermorphin, [l‐Tic2]dermorphin carboxylic acid and [d‐Tic2]dermorphin: all [l‐Tic2]peptides were converted from agonists to δ‐selective antagonists. The NMR conformational study in a dimethylsulfoxide/water cryoprotective mixture at low temperature shows diagnostic side‐chainside‐chain NOEs in the spectra of all [l‐Tic2]peptides and hints that the 90° arrangement of the the two aromatic rings found in the cis‐Tyr‐l‐Tic moiety, typical of N ‐methyl naltrindole and other δ‐selective opiate antagonists, is responsible for the antagonist activity of all these peptides.
Permeabilization of the Mitochondrial Inner Membrane by Short Cecropin‐A–Melittin Hybrid Peptides
Authors: Díaz‐Achirica Pilar , Prieto Susana , Ubach Josep , Andreu David , Rial Eduardo , Rivas Luis
Published: March 2005

A number of cecropin‐A–melittin hybrid peptides have previously been shown to be potent antibacterial agents [Andreu, D., Ubach, J., Boman, A., Wahlin, B., Wade, D., Merrifield, R. B. & Boman, H. G. (1992) FEBS Lett. 296, 190–1941. In the present report we analyze their action on biological systems using rat liver mitochondria as a test system. We demonstrate that the longest peptide, cecropin‐A‐(1–8)‐melittin(l–18) permeabilizes the mitochondrial inner membrane allowing the movement of both charged and non‐charged solutes. Concentrations used have already been shown to be bactericidal. This effect is also demonstrated under respiring conditions where succinate oxidation is uncoupled. Shorter analogs also permeabilize mitochondria although at tenfold higher concentrations. Heparin potentiates the peptide effects at low concentrations, while at high concentration it becomes inhibitory. We propose that the cecropinmelittin analogs disrupt the mitochondrial membrane in a detergent‐like mode rather than by creating selective channels as had been previously suggested.
Activation of a GTP‐Binding Protein and a GTP‐Binding‐Protein‐Coupled Receptor Kinase (β‐Adrenergic‐Receptor Kinase‐1) by a Muscarinic Receptor m2 Mutant Lacking Phosphorylation Sites
Authors: Kameyama Kimihiko , Haga Kazuko , Haga Tatsuya , Moro Osamu , Sadée Wolfgang
Published: March 2005

A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by β‐adrenergic‐receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating β‐adrenergic‐receptor‐kinase‐1‐mediated phosphorylation of a glutathione S‐transferase fusion protein containing the m2 phosphorylation sites in an agonist‐dependent manner. Both mutant and wild‐type m2 receptors reconstituted with the guanine‐nucleotide‐binding regulatory proteins (G protein), Go and Gi2, displayed guanine‐nucleotide‐sensitive high‐affinity agonist binding, as assessed by displacement of [3H] quinuclidinylbenzilate binding with carbamoylcholine, and both stimulated guanosine 5′‐3‐O ‐[35S] thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H] quinuclidinyl‐benzilate binding were indistinguishable for the mutant and wild‐type m2 receptors. Moreover, the phosphorylation of the wild‐type m2 receptor by β‐adrenergic‐receptor kinase‐1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]qunuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of β‐adrenergic‐receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by β‐adrenergic‐receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.
The Amino Acid Sequence of the Red Kidney Bean Fe(III)‐Zn(II) Purple Acid Phosphatase
Authors: Klabunde Thomas , Stahl Bernd , Suerbaum Hildegard , Hahner Stephanie , Karas Michael , Hillenkamp Franz , Krebs Bernt , Witzel Herbert
Published: March 2005

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110–kDa glycoprotein with a Fe(III)‐Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)‐Fe(II) centers in monomeric 35–kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N‐glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen‐bromide‐generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met‐Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30–kDa methionine‐free fragment, the alignment of the Edman‐degraded tryptic peptides was obtained by MALDI‐MS analysis and enzymic microscale peptide laddering of overlapping Glu‐C‐generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.
Modulation of Activator Protein‐1 (AP‐1) Transcription Factor and Protein Kinase C by Hydrogen Peroxide and d‐α‐Tocopherol in Vascular Smooth Muscle Cells
Authors: Stäuble Barbara , Boscoboinik Daniel , Tasinato Andrea , Azzi Angelo
Published: March 2005

The effects of hydrogen peroxide, d‐α‐tocopherol and of d‐β‐tocopherol on proliferation, protein kinase C and activator protein‐1 (AP‐1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by d‐α‐tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, d‐α‐tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol‐myristate‐acetate‐stimulated AP‐1 binding to DNA but stimulated it if protein kinase C was down‐regulated or inhibited. d‐α‐Tocopherol promoted AP‐1 activation in quiescent cells but prevented its activation by phorbol myristate acetate.

None of the described effects of d‐α‐tocopherol were shared by d‐β‐tocopherol, suggesting a non‐antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and d‐α‐tocopherol affect more than one element in the cell signal‐transduction cascade.
Purification and Biochemical Characterization of TrwC, the Helicase Involved in Plasmid R388 Conjugal DNA Transfer
Authors: Grandoso Guadalupe , Llosa Matxalen , Zabala Juan Carlos , de la Cruz Fernando
Published: March 2005

TrwC is an essential protein in conjugative DNA transfer of the broad‐host‐range plasmid R388. TrwC was purified in two chromatographic steps from TrwC‐overproducing bacteria. The purification procedure resulted in >90% pure TrwC protein, which was free of contaminating nuclease activities. TrwC behaved as a dimer in gel‐filtration chromatography in the presence of 550 mM NaCl, and had a pi of 10.1. The purified protein showed in‐vitro ssDNA‐dependent nucleoside‐5′‐triphosphatase and DNA helicase activities. ATP was the preferred substrate for the NTP hydrolysis reaction, which required Mg2+. The helicase activity was dependent on ATP and Mg2+. The efficiency of the unwinding reaction catalyzed by TrwC ranged from >90% of fragment displaced for a 93–nucleotide sequence to <5% for a 365–nucleotide sequence. Unwinding was unidirectional in the 5′ to 3′ direction. The enzyme turned over very slowly from one DNA substrate molecule to another. TrwC is only the second DNA helicase to be described which is involved in conjugative DNA transfer. The biochemical properties of TrwC described here confirm its functional relatedness to helicase I (Tral) encoded by plasmid F of E. coli.
Effects of the Tyr64 Substitution on the Stability of Cytochrome c553, a low Oxidoreduction‐Potential Cytochrome from Desulfovibrio vulgaris Hildenborough
Authors: Blanchard Laurence , Dolla Alain , Bersch Beate , Forest Eric , Bianco Pierre , Wall Judy , Marion Dominique , Guerlesquin Françoise
Published: March 2005

Cytochrome c553 from sulfate‐reducing bacteria is a lowoxidoreduction‐potential cytochrome. The primary and tertiary structures show notable differences when compared to mitochondrial cytochromes. Tyr64 replacement in cytochrome c553 provides evidence that this residue is not directly involved in the potential modulation but is mostly implicated in the hydrogenbond network around the heme. While the different variants obtained did not induce drastic structural modifications, they did affect the stability of the protein. This decrease of stability in acidic and alkaline environments was observed by variations in the optical spectra and by mass spectrometry. In addition, the mobility of aromatic side‐chain was found to be increased in the mutant proteins as monitored by two‐dimensional NMR spectroscopy.
Reactions of Prostaglandin H Synthase with Monosubstituted Hydrazines and Diazenes
Authors: Mahy Jean‐Pierre , Gaspard Sarra , Delaforge Marcel , Mansuy Daniel
Published: March 2005

The reaction of p‐chlorophenylhydrazine with prostaglandin H synthase (PGHS) Fe(III) under aerobic conditions leads to a partial destruction of the heme and to a new complex absorbing at 436 nm. This complex is also obtained by reaction of p‐chlorophenyldiazene (pClPhN = NH) with PGHS Fe(III) under anaerobic conditions and by oxidation of the PGHS Fe(II)(pClPhN = NH) diazene complex by Fe(CN)6K3. The similarity between those reactions and those of arylhydrazines and aryldiazenes with other hemoproteins such as cytochrome P450 and hemoglobin and myoglobin, as well as the similarities between the spectroscopic and chemical properties of this complex and those of the σ‐aryl complexes of other hemoproteins such as hemoglobin and myoglobin, strongly suggested a PGHS Fe(III)‐pClPh structure for this complex. It was completely established after the extraction of its heme, by butan‐2‐one at 0°C under neutral or acidic conditions, which led to the σ‐aryl PGHS‐Fe(III)‐pClPh complex and to N‐phenylprotoporphyrin IX, respectively. A mechanism is proposed for the formation of the PGHS Fe(III) pClPh complex; it includes the reduction of PGHS Fe(III) into PGHS Fe(II) with formation of the diazene pClPhN = NH. This diazene can bind to PGHS Fe(II) or be oxidized with formation of pClPh free radicals. These radicals can react with PGHS Fe(II) to form the PGHS Fe(III)‐pClPh complex or with the protein, or may initiate free radical oxidations which could lead to destruction of the heme or of the protein.

Other alkylhydrazines or arylhydrazines also react with PGHS Fe(III) under aerobic conditions with the formation of PGHS Fe(III)‐R or aryl (Ar) complexes and heme destruction. Alkylhydrazines such as methylhydrazine, which lead to very reactive alkyl radicals, lead to very low amounts of PGHS Fe(III)‐R complex and high amounts of heme destruction, whereas arylhydrazines bearing electron‐withdrawing substituents such as 3,4‐dichlorophenylhydrazine, which lead to stabilized aryl radicals, lead to a high amounts of PGHS Fe(III)‐Ar complex and low amounts of heme destruction.

Some information concerning the structure of the active site of PGHS has been drawn from those studies as follows: (a) the formation of σ‐aryl PGHS Fe(III)‐Ar and PGHS Fe(II)(ArN = NH) complexes bearing bulky ligands on the iron is in agreement with a large hydrophobic active site with an easily accessible iron atom and, (b) a comparison of the UV‐visible characteristics of PGHS Fe(III)‐R or Ar complexes with those of other hemoprotein‐Fe(III)‐R or hemoprotein‐Fe(III)‐Ar complexes is in agreement with a histidine as the axial proximal ligand of the iron of PGHS.

Finally, the formation of iron(III)‐R or iron (III)‐Ar complexes and the free radical oxidative degradation of the heme could both explain why monosubstituted hydrazines are powerful inhibitors of PGHS and very bad reducing cosubstrates of the peroxidase function of PGHS.
Structural Determinants of Substrate Selection by the Human Insulin‐Receptor Protein‐Tyrosine Kinase
Authors: Keane Noeleen E. , Chavanieu Alain , Quirk Philip G. , Evans James S. , Levine Barry A. , Calas Bernard , Wei Lei , Ellis Leland
Published: March 2005

Using NMR spectroscopy to visualise tyrosine phosphorylation kinetics in real time, we have investigated the sequence‐dependent determinants of the selectivity of the human insulin receptor protein‐tyrosine kinase for different tyrosine residues. The peptides used encompass the multipletyrosine‐containing autophosphorylation site sequences from the insulin receptor kinase core domain (Tyr1158, Tyr162 and Tyr1163) and from its specific C‐terminal tail domain (Tyr1328 and Tyr1334). Comparison of the phosphorylation kinetics with those found for the tyrosine residues on a peptide comprising the regulatory tyrosine phosphorylation site of cdc2 points to the role of the primary sequence context of the phosphate acceptor. The particularly deleterious influence of a basic residue immediately C‐terminal to the tyrosine is discussed in relation to the autophosphorylation properties of the regulatory loop regions of the insulin and epidermal growth factor receptor kinases. The data further suggest that receptor tyrosine kinase active sites and their substrate targets act in concert to ensure that specific downstream effects are activated.
Ca2+ Binding to Cardiac Troponin C in the Myofilament Lattice and Its Relation to the Myofibrillar ATPase Activity
Authors: Morimoto Sachio , Ohtsuki Iwao
Published: March 2005

The Ca2+‐binding properties of troponin C in the intact myofilament lattice and their relation to the activation of ATPase were investigated with isolated porcine cardiac myofibrils. Ca2+ binding, which is composed of two classes of binding sites with different affinities (classes 1 and 2), was clearly detected by a novel method for subtracting the large background activity of myofibrillar Ca2+ binding. The classes 1 and 2 were equivalent stoichiometrically to the two high‐affinity sites (sites III and IV) and a single low‐affinity site (site II) of troponin C. In the presence of ATP, positive cooperativity was observed in the Ca2+ binding of class‐2 sites and the Hill equation parameters were in excellent agreement with those for the Ca2+‐activated myofibrillar ATPase activity, which indicated that the activation of ATPase is a linear function of the Ca2+ occupancy of site II. In the absence of ATP, a marked increase in the affinity of only class‐2 sites was observed while the cooperativity was lost. These results provide direct evidence that some feedback mechanism exists between myosin crossbridge attachment and the Ca2+ binding to site II of troponin C, which may thus confer positive cooperativity on the Ca2+ activation of myofibrillar ATPase activity.
Effects of Glucose on the Activation and Translocation of Glycogen Synthase in Diabetic Rat Hepatocytes
Authors: Fernández‐Novell Josep M. , Ariño Joaquín , Guinovart Joan J.
Published: March 2005

Incubation of hepatocytes from diabetic rats with glucose results in the translocation of glycogen synthase from soluble fractions to fractions which sediment at 9200 g. The extent of the translocation correlates positively with the intracellular concentration of glucose 6‐phosphate. No difference was found between healthy and diabetic rats in the capacity of glycogen synthase to translocate to pellets in response to an increase in glucose 6‐phosphate. In diabetic hepatocytes, glycogen synthase in the supernatant fractions was not activated upon incubation of the cells with glucose, whereas this sugar was able to activate the enzyme found in the fractions that could be pelleted. In the 9200‐g pellets, the glycogen synthase activity ratio (–glucose 6‐phosphate/+glucose 6‐phosphate) from both healthy and diabetic animals correlated with the intracellular glucose 6‐phosphate levels. In the supernatants, the glycogen synthase activity ratio from healthy cells also correlated with glucose 6‐phosphate levels. In contrast, in diabetic cells the activation state of the soluble enzyme remained essentially unchanged despite the accumulation of glucose 6‐phosphate.
Flies in the Group Cyclorrhapha Use (3S)‐3‐Hydroxyretinal as a Unique Visual Pigment Chromophore
Authors: Seki Takaharu , Isono Kunio , Ito Masayoshi , Katsuta Yuko
Published: March 2005

In the class Insecta, retinal and 3‐hydroxyretinal are used as chromophores of visual pigments, but the absolute structure of the 3‐hydroxyretinal chromophore has yet to be clarified. This study investigates the chirality of 3‐hydroxyretinal in the compound eyes of five representative orders of insects. In the orders Odonata, Hemiptera, Neuroptera and Lepidoptera, and suborders Nematocera and Brachycera of the Diptera, only (3R)‐3‐hydroxyretinal isomers were detected, but dipterans of the suborder Cyclorrhapha (higher flies) had the (3S)‐11‐cis enantiomer and a mixture of (3R)‐all‐trans and S(3S)‐all‐trans 3‐hydroxyretinal enantiomers; the ratio of the (3R) enantiomer to the sum of both enantiomers of the all‐trans isomer was in the range 9–32%. Despite differences in feeding habits, including one species that is a butterfly parasite, all higher flies analysed to date share the same pattern of 3‐hydroxyretinal enantiomers, making them a unique group with regard to the nature of the visual pigment chromophore.
Linoleic Acid Transport by Human Placental Syncytiotrophoblast Membranes
Authors: Lafond Julie , Simoneau Lucie , Savard Roland , Gagnon Marie‐Claude
Published: March 2005

The placenta syncytiotrophoblast is the site of exchange of nutrients, lipids and minerals between the mother and the fetus. In order to characterize the transport of fatty acids by the placenta, we purified bipolar syncytiotrophoblast brush border and basal plasma membranes from human placenta. These purified brush border and basal plasma membranes enriched 3‐fold and 22‐fold, respectively, in sodium/potassium‐ATPase and 27‐fold and 6‐fold in alkaline phosphatase activity, compared with the placental homogenates. Fatty acid transport was performed at different fatty acid/albumin ratios to evaluate the optimal uptake conditions. The maximal transport efficiency, for linoleic acid bound to albumin by sonication, was obtained with a 6:1 fatty acid/albumin ratio in brush border and basal plasma membranes. The linoleic acid transport observed with brush border membranes followed Michaelis‐Menten kinetics, with a Michaelis constant of 7.89±0.01 μM and a maximal incorporation rate of 30.80±6.39 pmol · g−1· min−1. Linoleic acid transport was very low in basal plasma membranes and we obtained a Michaelis constant of 0.95±0.01 μM and a maximal incorporation rate of 1.62±5.06pmol · mg−1· min−1. In order to show that linoleic acid accumulated within brush border and plasma membrane vesicles, and to eliminate the possibility of a non‐specific binding of fatty acid to these membranes, we demonstrated by an osmolarity experiment, the decrease of the linoleic acid transport in brush border and basal plasma membranes obtained in the presence of 455 μM essential fatty acid at 23°C for 180 min. The results presented in this study suggest that linoleic acid is transported significantly by syncytiotrophoblast brush border membranes and basal plasma membranes. Thus, it may represent a unidirectional transport from mother to fetus through the brush border membranes facing the mother, followed by transport at a slower rate through basal plasma membranes facing the fetus.
The Mechanism of Dinitrogen Reduction by Molybdenum Nitrogenases
Authors: Leigh G. Jeffery
Published: March 2005

A mechanism for biological nitrogen fixation–Evidence for this proposal–A mechanism for biological nitrogen fixation – Conclusions
Turnover of Glutathione S‐Transferase α mRNAs is Accelerated by 12‐O‐Tetradecanoyl Phorbol‐13‐Acetate in Human Hepatoma and Colon Carcinoma Cell Lines
Authors: Eickelmann Peter , Morel Fabrice , Schulz Wolfgang A. , Sies Helmut
Published: March 2005

The phorbol ester, 12‐O ‐tetradecanoyl phorbol‐13‐acetate (TPA), known to induce murine glutathione S ‐transferase (GST) Ya, was examined for its effect on the expression of human GST α. Unexpectedly, 24‐h treatment of the human hepatoma cell line HepG2 with 100 nmol/1 TPA caused a decrease of the GST α mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c‐jun and jun‐B were elevated after 2 h. The decrease of GST α mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST α. In the presence of Actinomycin D, GST α mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST α mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST α protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST α expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.
Autoregulation of Actin Synthesis in Hepatocytes by Transcriptional and Posttranscriptional Mechanisms
Authors: Reuner Karl Heinrich , Wiederhold Matthias , Dunker Petra , Just Ingo , Bohle Rainer Maria , Kröger Manfred , Katz Norbert
Published: March 2005

Treatment of rat hepatocytes with the filamentous‐actin‐stabilizing toxin phalloidin decreased the amount of globular actin by 77% in the cytosol and by 80% in the nucleus within 12 h. Simultaneously, actin mRNA was specifically increased by 230%. The de‐novo synthesis of actin mRNA, as measured by nuclear run‐on transcription, was enhanced by 250%. Treatment of cells with actinomycin D blocked the increase of actin mRNA. The apparent half‐life of actin mRNA was not significantly altered during treatment with phalloidin.

In contrast, the globular‐actin‐stabilizing botulinum C2 toxin increased the amount of cytosolic globular actin by 50% within 12 h. Simultaneously, the actin mRNA level was decreased by 62%. However, de‐novo synthesis of actin mRNA was not impaired. The apparent half‐life of actin mRNA was decreased by approximately 60% during treatment with C2 toxin.

The data strongly suggest an autoregulatory control of actin synthesis on the basis of the globular/filamentous actin ratio in rat hepatocytes at the transcriptional as well as at the posttranscriptional levels.
Regulation of Mammalian Ornithine Decarboxylase
Authors: Lövkvist‐Wallström Eva , Stjernborg‐Ulvsbäck Louise , Scheffler Immo E. , Persson Lo
Published: March 2005

One of the cellular responses to hypotonic stress is a marked induction of a key regulatory enzyme in the polyamine biosynthetic pathway, i.e. ornithine decarboxylase (ODC). This increase in ODC activity appears to be a physiological response since the elevated putrescine production seen after the hypotonic shock renders the cells less sensitive to the decrease in osmolarity. In the present study, we have investigated the mechanisms by which the hypotonicity may induce ODC activity. We provide support for a translational mechanism, closely related to the polyamine‐mediated feedback regulation of ODC synthesis. In addition, we have examined whether the long G+C‐rich 5′ untranslated region of the ODC mRNA, which has been demonstrated to negatively affect the translatability of the message, is of any importance for the induction of ODC by hypotonic stress. Chinese hamster ovary (CHO) cells expressing ODC mRNA, with or without the 5′ untranslated region, were isolated after transfecting ODC‐deficient CHO cells with the appropriate constructs. Hypotonic treatment of the stable transfectants, however, revealed no major difference in ODC induction between the cells expressing a full‐length ODC mRNA and those expressing an ODC mRNA deleted of its 5′ untranslated region, demonstrating that this part of the message was not essential for the osmotic effects on ODC expression.
Inter‐Species DNA Polymerase δ Chimeras are Functional in Saccharomyces Cerevisiae
Authors: Moussy Geneviève , De Recondo Anne‐Marie , Baldacci Giuseppe
Published: March 2005

The catalytic subunits of DNA polymerase δ of Schizosaccharomyces pombe and Saccharomyces cerevisiae share over 50% identity. The capability of S. pombe DNA polymerase δ to complement two thermosensitive mutants of S. cerevisiae was studied in vivo and it was determined that complementation was allele dependent. However, DNA polymerase δ from S. pombe did not restore growth of a S. cerevisiae strain containing a disrupted chromosomal copy of the POL3 gene that encodes DNA polymerase δ. To identify the regions of DNA polymerase δ responsible for species‐specific interactions, we constructed different chimeras with S. cerevisiae and S. pombe DNA polymerase δ genes. The growth of a S. cerevisiae strain with a disrupted chromosomal POL3 gene was studied after transformation with plasmids expressing different chimeras. A 1254‐bp region located in the 3′ region of the S. cerevisiae POL3 gene is responsible for species‐specific functions.
The 14‐3‐3 Proteins Encoded by the BMH1 and BMH2 Genes are Essential in the Yeast Saccharomyces cerevisiae and Can be Replaced by a Plant Homologue
Authors: Van Heusden G. Paul H. , Griffiths Dominic J. F. , Ford Jon C. , Chin‐A‐Woeng Thomas F. C. , Schrader Peter A. T. , Carr Anthony M. , Steensma H. Yde
Published: March 2005

The 14‐3‐3 proteins comprise a family of highly conserved acidic proteins. Several activities have been ascribed to these proteins, including activation of tyrosine and tryptophan hydroxylases in the presence of calcium/calmodulin‐dependent protein kinase II, regulation of protein kinase C, phospholipase A2 activity, stimulation of exocytosis and activation of bacterial exoenzyme S (ExoS) during ADP‐ribosy‐lation of host proteins. In addition, a plant 14‐3‐3 protein is present in a G‐box DNA/protein‐binding complex. Previously, we isolated the BMH1 gene from Saccharomyces cerevisiae encoding a putative 14‐3‐3 protein. Using the polymerase chain reaction method, we have isolated a second yeast gene encoding a 14‐3‐3 protein (BMH2). While disruption of either BMH1 or BMH2 alone had little effect, it was impossible to obtain viable cells with both genes disrupted. The cDNA encoding a plant 14‐3‐3 protein under the control of the inducible GAL1 promoter complemented the double disruption. Transfer of the complemented double disruptant to a medium with glucose resulted in the appearance of a high percentage of large budded cells. After prolonged incubation, these cells became enlarged with irregular buds and chains of cells defective in cell‐cell separation became visible. These results suggest an essential role of the 14‐3‐3 proteins, possibly at a later stage of the yeast cell cycle.
Inversion of Lipase Stereospecificity for Fluorogenic Alkyldiacyl Glycerols
Authors: Zandonella Gerhild , Haalck Lutz , Spener Fritz , Faber Kurt , Paltauf Fritz , Hermetter Albin
Published: March 2005

We synthesized enantiomeric 1‐O‐alkyl‐2,3‐diacyl‐sn‐glycerol and 3‐O‐alkyl‐1,2‐diacyl‐sn‐glycerol containing pyrene as a fluorescent reporter and the trinitrophenylamino residue as a fluorescence quencher; both reporter groups were covalently bound to the ω end of the acyl chains at positions sn‐2 and sn‐3(1), respectively. The fluorescence of the intact substrate molecules was very low. Chemical or enzymic release of the fatty acyl chains lead to fluorescence dequenching. The rate of lipolysis could be measured from the time‐dependent increase in fluorescence intensity. We used the respective substrates for the continuous determination of activity and stereopreference of four different microbial lipases from Chromobacterium viscosum, Candida rugosa, Pseudomonas sp., Rhizopus arrhizus, as well as cutinase from Fusarium solani and lipoprotein lipase from bovine milk. The stereopreference of the lipases depended, in general, on how the substrate was solubilized in the reaction medium. All lipases under investigation preferentially hydrolysed the sn‐1 acyl ester bond, if the lipid analog was dispersed in albumin‐containing Tris/HCl buffer in the absence of detergent or organic solvent.

In mixtures of 1:1 (by vol.) water/ethanol, the enzymes showed higher activity toward the sn‐3 acyl ester bond, except for lipoprotein lipase which preferred the sn‐1 acyl isomer under all conditions tested. Different stereopreferences were observed with the different lipases if the substrate was solubilized by amphiphiles (micelles of N‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonate). C. rugosa lipase and F. solani cutinase showed high stereopreference for the sn‐3 acyl ester, whereas Pseudomonas sp. lipase and C. viscosum lipase hydrolysed both enantiomers at similar rates.

From spectroscopic studies, it can be inferred that the conformation of the fluorescent lipids is probably similar in water, mixtures of water and organic solvents, and in micelles. The possible effects of reaction conditions on substrate accessibility and enzyme conformation on stereoselectivity of the respective lipases are discussed.
Hen‐Egg‐White Lysozyme Modified with Histamine
Authors: Fukamizo Tamo , Hatta Tomohisa , Goto Sachio
Published: March 2005

The chemical modification of Asp101 which is located at the upper end‐most site (site A) of the binding cleft of hen egg white lysozyme affects the sugar residue binding of the midmost site (site C) in addition to that of site A, and results in the considerable decrease in the enzymic activity [Fukamizo, T., Hayashi, K. & Goto, S. (1986) Eur. J. Biochem. 158, 463–467]. In the present study, Asp101 was modified with histamine and converted to [2‐imidazol‐4(5)‐ylethyl]asparagine. Contrary to the findings described above, the specific activity of the modified lysozyme was higher than that of the native lysozyme by a factor of about two, and the loss of sugar residue binding ability caused by the modification was found to be restricted to site A. From the H‐NMR spectra of the modified lysozyme, the pKa value of the imidazolylethyl group covalently attached to Asp101 was 7.1, and was higher than that of N‐acetylhistidinemethylamide (6.65). This indicates that the imidazolylethyl moiety is not exposed to the solvent but adheres to the surface of the lysozyme molecule in an unidentified manner. When N‐acetylglucosamine trisaccharide [(GlcNAc)3] was added to the modified lysozyme, the 1H‐NMR signals of H2 and H4 of the imidazolylethyl group were strongly affected. This indicates that the imidazolylethyl moiety is located near (GlcNAc)3 binding region. When the Hγ signal of Ile98 was saturated, nuclear Overhauser effects were observed on H2 and H4 resonances of the imidazolylethyl moiety. NOE was also observed on the signal of Trp63 H6 upon the saturation of the H4 signal of the imidazolylethyl moiety. Thus, the imidazolylethyl moiety should be located near Trp63 and Ile98, which are in the hydrophobic box most proximal to the sugar binding cleft. This situation of the imidazolylethyl moiety did not result in steric hindrance to the sugar residue binding at sites B and C. The modification affected only the sugar residue binding at site A, and resulted in the enhanced activity.
Identification of a Novel Amide Peptide, GLTPNMNSLFF‐NH2, Involved in the Control of Vas Deferens Motility in Lymnaea stagnalis
Authors: Li Ka Wan , El Filali Zineb , Van Golen Floral A. , Geraerts Wijnand P. M.
Published: March 2005

Peptide messengers are crucial for the execution of male copulatory processes in the snail Lymnaea stagnalis. In this study, we report the purification of a novel peptide from the penis complex of L. stagnalis. This peptide enhances the contraction frequency and contraction amplitude of the vas deferens. Amino acid sequencing and mass spectrometry indicate that the primary structure is GLTPNMNSLFF‐NH2. We propose that this amide peptide modulates the transfer rate of semen in the vas deferens.
Over‐production, Purification and Properties of the Uridine‐diphosphate‐N ‐Acetylmuramate: l‐alanine Ligase from Escherichia coli
Authors: Liger Dominique , Masson Anne , Blanot Didier , Van Heijenoort Jean , Parquet Claudine
Published: March 2005

The UDP‐N ‐acetylmuramate:l‐alanine ligase of Escherichia coli was over‐produced in strains harbouring recombinant plasmids bearing the murC gene under the control of the lac or trc promoter. Plasmid pAM1005, in which the promoter and ribosome‐binding site region of murC were removed and in which the gene was directly under the control of promoter trc, led to a 2000‐fold amplification of the l‐alanine‐adding activity after induction by isopropyl‐thio‐β‐D‐galactopyranoside. The murC gene product was visualized as a 50‐kDa protein accounting for approximately 50% of the cell protein. A two‐step purification led to 1 g of a homogeneous protein from an 18–1 culture. The N‐terminal sequence of the purified protein correlated with the nucleotide sequence of the murC gene. The presence of 2‐mercaptoethanol and glycerol was essential for the stability of the enzyme. The Km values for UDP‐TV‐acetylmuramic acid, l‐alanine and ATP/Mg2+ were estimated at 100, 20 and 450 μM, respectively. Under the optimal in vitro conditions a turnover number of 928 min−1 was calculated and a copy number/cell of 600 could be roughly estimated. The specificity of the enzyme for its substrates was investigated with various analogues. The enzyme also catalysed the reverse reaction.
Distinct Tissue Distribution in Pigs of Tenascin‐X and Tenascin‐C Transcripts
Authors: Geffrotin Claudine , Garrido Juan Jose , Tremet Loïc , Vaiman Marcel
Published: March 2005

Tenascin‐X and tenascin‐C glycoproteins are phylogenetically conserved components of the extracellular matrix, although their specific roles remain to be determined. cDNA probes were produced from pig tenascin‐X and tenascin‐C genes and were used to examine the tissue distribution of the transcripts in 28 tissues from Large‐White pigs, 4.5–42‐months old (called adults) and 17 tissues from 87‐day‐old fetuses. The hybridization of Northern blots with tenascin‐X probes revealed, in most tissues, a complex pattern of bands including a major band of about 13 kb, assumed to correspond to the main tenascin‐X transcript. Hybridization with the tenascin‐C probe showed two transcripts of 6.8 kb and 8.2 kb. The data from the ribonuclease‐protection technique showed that both genes displayed large variations in the transcription levels among the tissues analysed. Overall, the tenascin‐X gene was significantly expressed in two thirds of the tissues, and the tenascin‐C gene in about 50% of them. The highest tenascin‐X signals were observed in tendons, ligaments and, unexpectedly, in peripheral nerves. Other tissues, including colon, dermis, skin, heart, uterus, stomach, jejunum, placentae, aorta, lung, mammary and adrenal glands also exhibited significant signal intensities. In fetuses, mainly testes and skeletal muscle showed higher transcription levels than the adult counterparts. The tenascin‐C gene was predominantly transcribed in the ligament, tendon, adrenal gland and colon, and more weakly in the stomach, jejunum, lung and spinal cord. In fetuses, the tenascin‐C signal in the brain was higher than the signal in the brain of adult, whereas the reverse was true for the adrenal gland and the colon. Within a given tissue, the level of tenascin‐X and tenascin‐C transcripts varied greatly, indicating independent tenascin‐X and tenascin‐C transcription regulation mechanisms; this was particularly obvious in adult and fetal nerves but also in the dermis, skin, heart, uterus, placentae and aorta, where tenascin‐X RNA molecules were much more abundant than those of tenascin‐C. In addition, similar differences were observed in the skeletal muscle and adrenal gland of fetuses. In contrast, the amount of tenascin‐C transcripts in the fetal brain and adult spinal cord was higher than those for tenascin‐X. Our results draw attention to a possible specific role of tenascin‐X in the peripheral nerve physiology.
Maize DNA Polymerase 2 is a Phosphoprotein with Increasing Activity During Germination
Authors: Coello Patricia , Vázquez‐Ramos Jorge M.
Published: March 2005

DNA replication is a late event during maize germination and DNA polymerase activity increases as germination proceeds. A replicative α‐type DNA polymerase has been purified from maize seeds (DNA polymerase 2) and has been shown to be a multisubunit complex [Coello, P., Rodríguez, R., García, E. & Vázquez‐Ramos, J. M. (1992) Plant Mol. Biol. 20, 1159–1168; Coello, P., García, E. & Vázquez‐Ramos, J. M. (1994) Can. J. Botany 72, 818–822]. DNA polymerase 2 activity increased several fold during maize germination, with no apparent change in either the amount of holoenzyme or in any of the individual subunits. However, the level of phosphorylation of the 90‐, 70‐, 55‐ and 45‐kDa polypeptides changed in a cyclic fashion with their highest levels occurring at 11–14 h and 45–48 h of germination. Phosphate incorporated into the different polypeptides in the 11–14‐h period remained stable for at least the next 10 h (to 24 h of germination), the period of maximal enzyme activity. However, DNA polymerase 2 processivity was very similar in freshly prepared 3‐h and 24‐h enzymes, and no evidence was found that polymerase activity was modified by in vitro phosphorylation. The significance of these results is discussed.
Synthesis of a New Photoaffinity Probe, 5‐Azido‐[32P]UDPxylose, by UDPglucuronate Carboxylyase from Wheat Germ
Authors: Kyossev Zhetcho N. , Drake Richard R. , Kyosseva Svetlana V. , Elbein Alan D.
Published: March 2005

The enzyme, UDPglucuronic acid carboxylyase (EC 4.1.1.35), was extensively purified from wheat germ, and was used to convert 5‐azido‐[32P]UDPglucuronic acid to 5‐azido‐[32P]UDPxylose, for use as a new photoaffinity probe. The carboxylyase was purified approximately 1200‐fold using conventional methods, and the enzyme preparation, at the final stage of purification, was stable to storage at −20°C for at least 9 months with little or no loss of activity. The partially purified carboxylyase catalyzed the conversion of 5‐azido‐[32P]UDPglucuronic acid to 5‐azido‐[32P]UDPxylose in good yield, and the UDPxylose probe was purified by ion‐exchange chromatography, and characterized. The newly synthesized photoaffinity analog, 5‐azido‐[32P]UDPxylose, should be a valuable tool in the purification of various xylosyltransferases.