bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that Adenine Nucleotides Modulate Nucleoside‐Transporter Function
Authors: Delicado Esmerilda G. , Casillas Teresa , Sen Raquel P. , Miras‐Portugal María Teresa
Published: March 2005

Uridine transport was investigated in cultured chromaffin cells and plasma membrane vesicles from chromaffin tissue. In intact cells, the kinetic parameters for uridine uptake were Km 150 ± 45 μM, and Vmax 414 ± 17 pmol · 106 cells−1· min−1. This low affinity for uridine and its inhibition by low concentrations of nitrobenzylthioinosine (Ki 3 nM) and dipyridamole (Ki 54 nM) are consistent with a facilitated diffusion nucleoside transport system. The IC50 value for the adenosine transport inhibition by uridine was very high (240 μM), agreeing with the relative affinities of these nucleosides in the chromaffin cell nucleoside transport system, which was 150‐fold higher for adenosine than for uridine. Uridine was significantly metabolized in chromaffin cells but not in plasma membrane vesicles. The affinity of uridine transport measured in these membrane vesicles was reproducible and similar to the affinity found for intact cells with a Km value of 185 ± 11 μM and a Vmax value of 4.24 ± 0.10 pmol · mg protein−1· s−1. These membrane preparations were employed to investigate the regulatory action of ATP and other nucleotide analogues on nucleoside transport. ATP increased the Vmax value but the Km value was not significantly modified. Adenosine 5′‐[β,γ‐imino]triphosphate, 1,N6‐ethenoadenosine 5′‐triphosphate, and adenosine(5′)‐tetraphospho(5′)adenosine (Ap4A) at 100 μM were able to mimic the ATP effect. These results agree with a regulatory role of ATP, and the uridine transport on chromaffin plasma membrane vesicles is a good model for analyzing the nucleoside‐transporter function and regulation.
A New Structural Transition of Serum Albumin Dependent on the State of Cys34
Authors: Christodoulou John , Sadler Peter J. , Tucker Alan
Published: March 2005

Reactions of fatty‐acid‐free bovine serum albumin and recombinant human albumin with a range of antiarthritic gold(I) complexes [auranofin, deacetylated auranofin, triethylphosphinegold(I) chloride] and related thiols (thioglucose, tetraacetylthioglucose, glutathione, dithiothreitol) have been investigated using 1H‐NMR spectroscopy.

In reactions of albumin with auranofin, tetraacetylthioglucose and dithiothreitol, release of cystine was detected, whereas for deacetylated auranofin, thioglucose and glutathione, mixed disulphides with cysteine were produced. It has been previously proposed that Cys34 of human and bovine serum albumins is partly blocked by disulphide formation with cysteine and glutathione. The above reactions lead to deblocking by thiol–disulphide interchange reactions. No release of glutathione from albumin was detected.

Changes in the His Hɛ1 regions of the 1H‐NMR spectra show that albumin exists in two structural forms dependent on whether the side‐chain of Cys34 is a free thiolate, or blocked by gold(I)triethylphosphine, by disulphide formation with cysteine or by another form of oxidation. We propose that Cys34 is either in a buried or in an exposed environment; the possible molecular basis of the structural change is discussed.

The relationship between reactions at Cys34, cysteine release, and the observed structural transition are discussed in terms of chrysotherapy, albumin metabolism and the use of gold(I) as a heavy atom derivative in X‐ray crystallographic studies of albumins.
The Function of Arginine 363 as the Substrate carboxyl‐binding Site in Escherichia coli Serine Hydroxymethyltransferase
Authors: Fratte Sonia Delle , Iurescia Sandra , Angelaccio Sebastiana , Bossa Francesco , Schirch Verne
Published: March 2005

Both the highly conserved Arg363 and Arg372 residues of Escherichia coli serine hydroxymethyltransferase were changed to alanine and lysine residues. Each of the four mutant proteins were purified to homogeneity and characterized with respect to spectral properties of the enzyme‐bound pyridoxal phosphate and kinetic properties with substrates and substrate analogs. The R372A and R372 K mutant enzymes exhibited spectra and kinetic properties close to those of the wild‐type enzyme. The R363 K mutant enzyme exhibited only 0.03% of the catalytic activity of the wild‐type enzyme and a 15‐fold reduction in affinity for glycine and serine. The R363A mutant enzyme did not bind serine and glycine and showed no activity with serine as the substrate. Both R363 K and R363A enzymes bound amino acid esters at the active site and catalyzed the retro‐aldol cleavage of serine ethyl ester and serinamide. The catalytic activity of the R363 K and R363A enzymes with the serine ethyl ester were about 0.006% and 0.1% of wild‐type enzyme activity with serine, respectively. The R363A mutant enzyme catalyzed the half transamination of d‐alanine methyl ester and l‐alanine methyl ester at rates similar to the rates of transamination of d‐alanine and l‐alanine by the wild‐type enzyme. The results are interpreted to show that R363 is the binding site of the amino acid substrate carboxyl group and that forming an ion pair between R363 and the substrate carboxyl group is an important feature in catalysis by serine hydroxymethyltransferase. Evidence is also provided that R363 may play a role in the substrate‐induced open to closed conformational change of the active site.
Production, Inhibitory Activity, Folding and Conformational Analysis of an N‐terminal and an Internal Deletion Variant of Chicken Cystatin
Authors: Auerswald Ennes A. , Nägler Dorit K. , Schulze Andreas J. , Engh Richard A. , Genenger Gabriele , Machleidt Werner , Fritz Hans
Published: March 2005

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg‐Glu‐Phe‐[Met1, IIe29, Leu89]‐chicken egg white cystatin gene in Escherichia coli. The variant des‐Ser1‐Pro11‐[Ala12, Glu13, Phe14, Met15, IIe29, Leu89]‐chicken cystatin (N‐del 2) and the variant Arg‐Glu‐Phe‐[Met1, IIe29]‐des‐Cys71‐Met89‐chicken cystatin (del‐helix II) were purified and characterized by inibition kinetics, far‐ultraviolet‐CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del‐helix II variant, shortened by 19 amino acids, is a basic, stefin‐like mini‐cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del‐helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far‐ultraviolet‐CD. With increasing Gdn/HCl concentration, a stabilization of secondary‐structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N‐del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000–100000‐fold higher than those obtained with natural and recombinant chicken cystatin.
A Diversified Family of 12‐kDa Proteins with a High Amino Acid Sequence Similarity to Macrophage Migration‐Inhibitory Factor (MIF)
Authors: Galat Andrzej , Rivière Sylvie , Bouet Françoise , Ménez André
Published: March 2005

Two isoforms of a bovine‐brain‐derived 12‐kDa protein (designated p12a and p12b) whose N‐termini have a high amino acid sequence similarity with the glycosylation‐inhibiting factor (GIF) and macrophage migration‐inhibitory factor (MIF) were purified to homogeneity. The complete amino acid sequence of bovine p12a (pI 9.5) was determined by Edman degradation of the intact molecule and overlapping fragments generated by proteolytic cleavage. The p12a isoform has nine and ten conservative substitutions versus human GIF (hGIF) and human MIF (hMIF), respectively. Molecular filtration revealed that both isoforms of p12 exist as monomers in aqueous solution. Circular dichroism spectra indicate that both isoforms of p12 consist of 39±3%α helix, 23±3%β structure and 15±3%β turns. Although the N‐terminal parts of p12a and p12b have weak amino acid sequence similarity with that of glutathione S‐transferase (GST) neither isoform of p12 was bound to a GST‐affinity gel nor had GST activity. Despite a high amino acid sequence similarity with human MIF neither of the p12 isoforms inhibited migration of the mouse monocyte‐macrophage cells P338D1.
Cytosolic Calcium Ions Regulate Lipid Mobility in the Plasma Membrane of the Human Megakaryoblastic Cell Line MEG‐01
Authors: Schooteureijer Annelies , Van Beekhuizen André E. , Tertoolen Leon G. J. , De Laat Siegfried W. , Akkerman Jan‐Willem N.
Published: March 2005

The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1′‐ditetradecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG‐01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53±0.06)×10−9 cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99±0.31)×10‐9 cm2/s and a mobile fraction of 58%. Stimulation with platelet‐activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D‐Phenylalanyl‐L‐prolyl‐L‐arginyl‐chloromethane‐treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.
Nitric Oxide Regulation of glyceraldehyde‐3‐phosphate Dehydrogenase Activity in Dictyostelium discoideum Cells and Lysates
Authors: Tao Yongping , Howlett Allyn , Klein Claudette
Published: March 2005

The ability of compounds releasing nitric oxide (NO) to regulate glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) activity was analysed both in cell homogenates and in intact Dictyostelium discoideum. The time course of GraPDH inactivation in cell lysates by NO‐releasing compounds suggests that two processes may be involved, one of which accounts for the majority of the inactivation and shows a close correlation with GraPDH ADP‐ribosylation. Maximal ADP‐ribosylation under these conditions exhibited a stoichiometry of about 0.4 mol ADP‐ribose/mol enzyme tetramer. NO‐mediated inhibition of GraPDH activity was attenuated if specific substrates, cofactors, or cysteine were added to cytosol preparations. Under such conditions, ADP‐ribosylation of the enzyme was correspondingly reduced or negligible. Intact cells treated with NO‐releasing compounds were shown to respond by rapidly decreasing their GraPDH activity. This inhibition was transient and, after a 10‐min incubation, enzyme activity returned to the level seen in control cells. The time course of these in vivo changes correlated well with those of the NO‐stimulated ADP‐ribosylation of GraPDH also seen in intact cells. The basis underlying the NO‐stimulated inhibition of GraPDH activity was investigated and found to reflect a decreased Vmax+ No changes in either the Km of the enzyme for its substrates or its state of polymerization were observed.
The Levels of Yeast Gluconeogenic mRNAs Respond to Environmental Factors
Authors: Mercado Juan J. , Smith Rachel , Sagliocco Francis A. , Brown Alistair J. P. , Gancedo Juana M.
Published: March 2005

The FBP1 and PCK1 genes encode the gluconeogenic enzymes fructose‐1,6‐bisphosphatase and phosphoenolpyruvate carboxykinase, respectively. In the yeast, Saccharomyces cerevisiae, the corresponding mRNAs are present at low levels during growth on glucose, but are present at elevated levels during growth on gluconeogenic carbon sources. We demonstrate that the levels of the FBP1 and PCK1 mRNAs are acutely sensitive to the addition of glucose to the medium and that the levels of these mRNAs decrease rapidly when glucose is added to the medium at a concentration of only 0.005%. At this concentration, glucose blocks FBP1 and PCK1 transcription, but has no effect on iso‐1 cytochrome c (CYCI) mRNA levels. Glucose also increases the rate of degradation of the PCK1 mRNA approximately twofold, but only has a slight effect upon FBP1 mRNA turnover. We show that the levels of the FBP1 and PCK1 mRNAs are also sensitive to other environmental factors. The levels of these mRNAs decrease transiently in response to a decrease of the pH from pH 7.5 to pH 6.5 in the medium, or to a mild temperature shock (from 24°C to 36°C). The latter response appears to be mediated by accelerated mRNA decay.
Cloning of Metalloprotease Genes in the Carpet Viper (Echis pyramidurm leakeyi)
Authors: Paine Mark J. I. , Moura‐Da‐Silva Ana M. , Theakston R. David G. , Crampton Julian M.
Published: March 2005

The genes coding for two metalloproteases, EcH‐I and EcH‐II, have been cloned from an Echis pyramidum leakeyi venom gland cDNA library. The cDNA sequences predict two zymogen molecules with strong amino acid sequence similarity and the same domain structure present in other members of the viper metalloprotease/disintegrin gene family. EcH‐I and EcH‐II contain pro‐protein, enzyme and disintegrin domains. Analysis of the cDNAs coding for EcH‐I, EcH‐II, jararhagin, trigramin and Ht‐e reveals a strong similarity, particularly in the untranslated regions and regions coding for the pro‐peptide. Comparison of EcH‐I and EcH‐II with venom metalloproteases, mammalian matrix‐degrading metalloproteases, sperm proteins, and a potential tumour suppressor gene highlights the presence of a number of motifs with potential functional significance.
Electron microscopy and biochemical characterization of a 350‐kDa annular hemolymph protein from the keyhole limpet Megathura crenulata
Authors: Harris J. Robin , Markl Jürgen
Published: March 2005

The isolation and biochemical characterization of an annular non‐hemocyanin hemolymph protein from a marine gastropod, the Californian giant keyhole limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of ‐SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring‐like structure with an approximately 2‐nm stain‐filled centre and, in another image projection, as a dimeric rectangular structure, approximately 8 nm × 14 nm. In deep stain, each of the components of the side‐on dimer also shows an indication of stain penetration within a central channel. Unlike the hemolymph protein limulin/C‐reactive protein from the horseshoe crab Limulus polyphemus, this Megathura protein does not possess any potential for the aggregation of phosphatidylcholine liposomes although it has an affinity for this phospholipid and causes bilayer destruction. It now appears likely that the previously reported lipid‐bilayer ionic‐conductance species in keyhole limpet hemolymph, attributed to hemocyanin, could indeed by due to the presence of this annular protein.
Rye Inhibitors of Animal α‐amylases Show Different Specifities, Aggregative Properties and IgE‐binding Capacities than Their Homologues from Wheat and Barley
Authors: García‐Casado Gloria , Sánchez‐Monge Rosa , López‐Otín Carlos , Salcedo Gabriel
Published: March 2005

Three new members of the α‐amylase/trypsin‐inhibitor family of cereal endosperm have been isolated from rye. N‐terminal amino acid sequence comparison revealed that two of the purified proteins were the rye homologues of the barley monomeric inhibitor (BMAI‐1) previously described, while the other rye protein corresponded to one of the subunits of the barley and wheat heterotetrameric inhibitors. However, the inhibitory specificities (active against human salivary α‐amylase), aggregative behaviours (mainly as dimeric forms) and IgE‐binding capacities (not recognized by sera from allergic patients) of the rye inhibitors were clearly different from those of their wheat and barley counterparts. These results indicate that homologous inhibitors may have distinctive properties in different cereal species.
Kex2p: a model for cellular endoprotease processing human immunodeficiency virus type 1 envelope glycoprotein precursor
Authors: Moulard Maxime , Achstetter Tilman , Kieny Marie‐Paule , Montagnier Luc , Bahraoui Elmostafa
Published: March 2005

The endoproteolytic cleavage of the envelope glycoprotein precursor (gp160) of the human immunodeficiency virus type 1 (HIV‐1) by a cellular protease is required for full activation of the virus. In this study, processing of gp160 was analyzed in vitro using the Kex2p endoprotease from the yeast Saccharomyces cerevisiae as a processing enzyme model. Endoproteolytic processing was examined using a synthetic peptide that mimics the cleavage site of HIV‐1 glycoprotein, and a recombinant gp160 bearing the entire sequence of the env gene product, including the conserved cleavage site Arg508‐Glu‐Lys‐Arg511. Coexpression in BHK‐21 of Kex2p and gp160 by recombinant vaccinia viruses demonstrates that Kex2p can correctly process the HIV‐1 glycoprotein to gp120 and gp41. Furthermore, recombinant gp160 and peptide were used as substrates and subjected to proteolysis with purified membranes from an S. cerevisiae strain overproducing the Kex2p endoprotease. Treatment of recombinant gp160, which has an apparent molecular mass of 127 kDa, with Kex2p and Western blot analysis showed that the precursor was cleaved into two products of about 101 and 34 kDa apparent molecular mass. Amino acid sequencing of the NH2‐terminus of the 34‐kDa product showed that the cleavage site of recombinant gp160 was between Arg511 and Ala512. Recombinant gp160 mutated at the sequence coding for the potential cleavage site, and mature recombinant gp120, however, were not cleaved when treated with Kex2p. In summary, our results show that Kex2p cleaves both the HIV‐1 envelope glycoprotein precursor and a synthetic peptide mimicking the cleavage site of HIV‐1 gp160 at the dibasic site, suggesting functional analogy between yeast Kex2p and the cellular protease responsible for the maturation of HIV‐1 envelope glycoproteins in infected human cells.
cDNA sequence and structure of a gene encoding trout testis high‐mobility‐group‐1 protein
Authors: Štros Michal , Nishikawa Sandra , Dixon Gordon H.
Published: March 2005

Perchloric acid extraction of trout testis nuclei revealed the presence of two large high‐mobility‐group (HMG) proteins, HMG‐T1 and HMG‐T2. The sequence of a complete cDNA (1407 bp) for trout testis HMG‐1 protein (referred as to HMG‐T1) has been determined. The deduced HMG‐T1 protein contains 203 amino acids with more than 86% similarity to mammalian HMG‐1 proteins. A single‐sized mRNA for HMG‐T1 has been detected by Northern‐blot analysis consistent with the size derived from the HMG‐T1 cDNA. Amplification of human and trout genomic DNAs by polymerase chain reaction using primers specific for trout and human HMG‐1 cDNAs revealed that unlike the human genome, which contains predominantly intronless HMG‐1 sequences, intronless HMG‐T1 sequences were not found in the fish genome. Southern‐blot analysis suggested that the trout testis HMG‐1 gene is encoded by at least two sequences with high similarity.

A gene encoding HMG‐T1 protein has been isolated from a trout testis genomic library and by PCR of trout genomic DNA (3879 bp); The trout testis HMG‐1 gene is organized into five exons (four exons corresponding to the protein‐coding region) and its exon/intron boundaries are identical to those of the human HMG‐2 gene [Shirakawa, H. & Yoshida, M. (1992). J. Biol. Chem. 267, 6641–6645] suggesting the evolution of HMG‐1 and HMG‐2 genes from a common ancestor.
Tyr394 and Tyr505 are Autophosphorylated in Recombinant Lck Protein‐tyrosine Kinase Expressed in Escherichia coli
Authors: Jullien Pascale , Bougeret Cécile , Camoin Luc , Bodeus Monique , Durand Hervé , Disanto James P. , Fischer Siegmund , Benarous Richard
Published: March 2005

The activity of the Src family protein‐tyrosine kinase P56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505. Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates P56lck activity.

To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S‐transferase expression system to express wild‐type Lck, the mutants [Y394F]Lck and [Y505F]Lck, a kinase‐deficient P56lck with a mutation of the ATP‐binding site [K273E]Lck and a double mutant [Y394F, Y505F]Lck. We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild‐type Lck both in vivo and in vitro.

Wild‐type Lck, [Y505F]Lck and [Y394F]Lck were phosphorylated on tyrosine. Both the kinase‐deficient mutant[K273E]Lck and the double mutant [Y394F, Y505F]Lck did not react with monoclonal anti‐phosphotyrosine antibody [anti‐Y(P) mAb], thus providing evidence that (a) the bacterial strains used lacked intrinsic protein‐tyrosine kinase activities, and therefore tyrosine phosphorylations of wild‐type Lck, [Y505F]Lck and [Y394F]Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505. Phosphopeptide mapping analysis confirmed that P56lck can undergo autophosphorylation on these two tyrosine residues. We propose that autophosphorylation at Tyr505 of P56lck may represent an accessory mechanism for the down‐regulation of the tyrosine kinase activity of P56lck.
The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide
Authors: Ramesh Vasudevan , Frederick Ronnie O. , Syed Shabih E. H. , Gibson Catherine F. , Yang Ji‐chun , Roberts Gordon C. K.
Published: March 2005

The effects of the binding of the corepressor l‐tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N‐enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the ‘helix‐turn‐helix’ DNA‐binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N‐terminal region of the protein and in the DNA‐binding domain, perhaps reflecting a conformational equilibrium.
Expression, assembly and secretion of a fully active plant ferredoxin‐NADP+ reductase by Saccharomyces cerevisiae
Authors: Ottado Jorgelina , Arakaki Adrián K. , Calcaterra Nora B. , Ceccarelli Eduardo A.
Published: March 2005

The flavoprotein ferredoxin‐NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin. Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library [Newman, B. J. & Gray, J. C. (1988) Plant Mol. Biol. 10, 511–520]. Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin‐NADP+ reductase to the chloroplast. These sequences were introduced into a yeast shuttle vector in frame with the mating factor α1 secretion‐signal coding region under the control of its natural mating factor α1 promoter. Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin‐NADP+ reductase. In both cases, a 35‐kDa polypeptide was the major product. N‐terminal sequencing of the secreted proteins indicates processing at position ‐1 with respect to the N‐terminus of the pea mature enzyme. Yeast cells transformed with plasmid encoding the ferredoxin‐NADP+ reductase precursor secrete four‐times more ferredoxin‐NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme. Ferredoxin‐NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin‐NADP+ reductase. The overall results indicate that pea ferredoxin‐NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion.
A High‐resolution 15N Solid‐state NMR Study of Collagen and Related Polypeptides
Authors: Naito Akira , Tuzi Satoru , Saitô Hazime
Published: March 2005

High‐resolution solid‐state 15N‐NMR was used to clarify the effect of hydration on the stability of the coiled‐coil triple‐helix conformation of Gly‐Xaa‐Yaa repeating units in collagen and the collagen‐like polypeptides, (Pro‐Ala‐Gly)n, and (Pro‐Pro‐Gly)10, because the stability is thought to be related to the presence of (Gly)NH…O=C(Xaa or Pro) hydrogen bonds. The 15N‐NMR signals of these samples were narrowed upon hydration, mainly due to hydration‐induced conformational change or rearrangement of the repeating units. In particular, the 15N chemical shifts of the Gly N‐H group and the high‐field (low‐frequency) shoulder peak of Pro nitrogen signals in (Pro‐Pro‐Gly)10 were shifted downfield (4.9 ppm and 6.8 ppm, respectively) with increasing relative humidity, while the corresponding peaks for collagen and (Pro‐Ala‐Gly)n, were unchanged and close to the 15N shift of (Pro‐Pro‐Gly)10, in the hydrated state. Such downfield shifts are consistent with the formation of N‐H…O=C hydrogen bonds. In agreement with the NMR results, it was found that the (Gly)NH…O=C (Xaa or Pro) hydrogen bonds are retained in dehydrated collagen fibrils but not in partially dehydrated (Pro‐Pro‐Gly)10. No evidence was obtained for the partial formation of the 31 helix form (Pro)n or (Gly)n either under hydrated or dehydrated conditions. It is concluded that the Gly 15N chemical shift is a very sensitive probe for studying supercoiling in collagen and collagen‐like polypeptides.
Genomic Cloning and Sequence Analyses of the Bovine α‐, βA‐ and βB‐Inhibin/activin Genes
Authors: Thompson Devon A. , Cronin Ciarán N. , Martin Finian
Published: March 2005

Inhibins and activins are dimeric peptide hormones that regulate the circulating levels of follicle‐stimulating hormone (FSH). In turn, FSH stimulates inhibin gene expression in the ovarian follicle; studies to date suggest that this effect is mediated by cAMP and that a cAMP‐responsive element, identified in the 5′‐flanking region of the α‐inhibin gene, at least partially effects this response. To explore further the transcriptional regulation of the inhibin/activin genes, we have isolated and sequenced the 5′‐flanking regions of the bovine α‐, βA‐ and βB‐inhibin/activin subunit genes and have analysed these regions by primer‐extension analysis and DNase I footprinting with the transcription factor AP‐2. Analyses indicated that all three gene promoter regions have a number of AP‐2‐binding sites that are resistant to competition by poly(dI‐dC), suggesting that cAMP may control the inhibin/activin ratio by operating through alternative signal‐transduction pathways or that inhibin/activin gene expression may be controlled by signals operating through the protein kinase C pathway. A comparison of the DNA sequences protected by AP‐2 against DNase I digestion revealed a consensus AP‐2‐binding site of 5′‐GSCCCDSS‐3′, where S represents a base pairing involving three (C or G) hydrogen bonds and D represents any base other than C. The nucleotide sequences of the bovine β‐subunit structural genes also are reported.
Enzymology of FK‐506 biosynthesis
Authors: Shafiee Ali , Motamedi Haideh , Chen Tom
Published: March 2005

FK‐506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope‐feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31‐O ‐desmethylFK‐506, a metabolic precursor for the biosynthesis of FK‐506, a S ‐adenosyl‐l‐methionine‐dependent enzyme assay was developed and the enzyme 31‐O ‐desmethylFK‐506 O:methyl‐transferase was isolated from an extract of Streptomyces sp. MA 6858 and purified to near homogeneity. 31‐O‐DesmethylFK‐506 O:methyltransferase is a monomeric protein with an apparent molecular mass of 30000 Da and a pI of 4.4. The first 38 N‐terminal amino acids have been sequenced and are H2N‐SDVVETLRLPNGATVAHVNAGEAQFLYREIFTDRXYLRH. Functionally, this enzyme has a requirement for Mg2+ with an optimum temperature of 34°C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31‐O ‐desmethylimmunomycin as efficiently as its own natural substrate, 31‐O ‐desmethylFK‐506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31‐O‐bis‐desmethyl‐, 15,31‐O‐bisdesmethyl‐, 13,15,31‐O‐trisdesmethyl‐ and 31‐O‐19,22‐cyclic‐hemiketalimmunomycins, which are all structural analogues of FK‐506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C‐32 position of the 31‐O‐desmethylFK‐506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31‐O ‐desmethyl‐immunomycin:O methyltransferase, previously isolated from a cell‐free extract of Streptomyces hygroscopicus var ascomyceticus, an immunomycin (ascomycin/FK‐520) producer.
Inhibition of Cyclin‐Dependent Kinases by Purine Analogues
Authors: Veselý Jaroslav , Havliček Libor , Strnad Miroslav , Blow J. Julian , Donella‐Deana Arianna , Pinna Lorenzo , Letham David S. , Kato Jun‐ya , Detivaud Lénaïck , Leclerc Sophie , Meijer Laurent
Published: March 2005

While testing purines related to the non‐specific protein kinase inhibitors N6‐dimethylaminopurine and N6‐(2‐isopentenyl)adenine as potential inhibitors of the p34cdc2/cyclin B kinase, we discovered a compound with high specificity, 2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non‐competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34cdk4/cyclin D1, p40cdk6/cyclin D3, CAMP‐dependent and cGMP‐dependent kinases, eight protein kinase C isoforms, calmodulin‐dependent kinase II, myosin light‐chain kinase, mitogen‐activated S6 kinase, casein kinase 2, double‐stranded RNA‐activated protein kinase, AMP‐stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell‐cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP‐kinase (and its starfish homologue p44mpk) are substantially inhibited by olomoucine (IC50 values are 7, 7, 7, 3 and 25 μM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC50 values greater than 1 mM and 150 μM, respectively). N6‐(2‐Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC50, values of 50–100 μM for many kinases. The purine specificity of cyclin‐dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9‐substituted purines exert a strong inhibitory effect on the p34cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain‐specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M‐phase‐promoting factor activity in metaphase‐arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti‐mitotic reagent that may preferentially inhibit certain steps of the cell cycle.
Mo(V) Electron Paramagnetic Resonance Signals from the Periplasmic Nitrate Reductase of Thiosphaera Pantotropha
Authors: Bennett Brian , Berks Ben C. , Ferguson Stuart J. , Thomson Andrew J. , Richardson David J.
Published: March 2005

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane‐bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrifcans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum‐containing reductases, molybdenum‐hydroxylase‐like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.
Generation by Limited Proteolysis of a Catalytically Active 39‐kDa Protein from the 115‐kDa form of Phosphatidylinositol‐glycan‐specific Phospholipase D from Bovine Serum
Authors: Heller Manfred , Bütikofer Peter , Brodbeck Urs
Published: March 2005

It has been suggested previously that small amounts of the mature 115‐kDa form of phosphatidylinositol (PtdIns)‐glycan‐specific phospholipase D from bovine serum may exist as a 47‐kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns‐glycan‐specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X‐100. Trypsin cleaved the 115‐kDa form of PtdIns‐glycan‐specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non‐denaturing conditions, the polypeptides remained tightly but non‐covalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion‐exchange chromatography. After renaturation, PtdIns‐glycan‐specific phospholipase D activity was found to be associated with a 39‐kDa fragment. Based on its size and its amino acid sequence, the active‐site‐containing fragment consisted of approximately 275 residues of the N‐terminal region of PtdIns‐glycan‐specific phospholipase D. The active 39‐kDa fragment hydrolyzed the PtdIns‐glycan‐anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane‐associated acetylcholinesterase and PtdIns.
Purification and Properties of Zinc‐metallophospholipase C from Pseudomonas fluorescens
Authors: Crevel Isabelle , Sally U , Carne Alexander , Katan Matilda
Published: March 2005

Phospholipase C produced by Pseudomonas fluorescens, isolated as a laboratory contaminant, has been purified to apparent homogeneity by ammonium sulphate fractionation, anion‐exchange and size‐exclusion chromatographies. The apparent molecular mass of the purified polypeptide was 39.5 kDa. Purified preparations of phospholipase C were used to characterize its enzymic properties and to obtain amino acid sequence of the N‐terminus of the molecule. The P. fluorescens phospholipase C hydrolysed PtdEtn, PtdCho and PtdSer (PtdEtn > PtdCho ≫ PtdSer) and was relatively thermostable. The enzyme was inactivated in the presence of chelating agent o‐phenanthroline and the activity restored after addition of zinc. Properties of this enzyme and in particular the requirement for zinc ions for the activity, revealed similarity with the well characterised Bacillus cereus phospholipase C. Similarities with other bacterial and mammalian enzymes reported to be related to the B. cereus type are discussed.
Purification and Properties of a Succinyltransferase from Pseudomonas aeruginosa Specific for both Arginine and Ornithine
Authors: Tricot Catherine , Vander Wauven Corinne , Wattiez Ruddy , Falmagne Paul , Stalon Victor
Published: March 2005

The arginine and ornithine succinyltransferase from Pseudomonas aeruginosa, a bifunctional enzyme involved in the aerobic utilization of arginine and ornithine, has been purified to homogeneity. The apparent molecular mass of the native enzyme was 150 kDa by gel filtration and 140 kDa by polyacrylamide gel electrophoresis under non‐denaturing conditions. After SDS/PAGE two subunits of 35 kDa and 37 kDa were evident, indicating that the enzyme is a heterotetramer. Microsequence analysis of the electroblotted protein bands gave two different but well‐conserved N‐terminal amino acid sequences.

The l‐arginine saturation curve followed Henri‐Michaelis kinetics with an apparent Km value of 0.5 mM. The sigmoidal saturation curve for l‐ornithine indicated allosteric behaviour. d‐Arginine, a competitive inhibitor with respect to l‐arginine, reduced l‐ornithine cooperativity. In the presence of spermidine, the l‐ornithine saturation curve became increasingly sigmoidal, the Hill coefficient shifting from 2.5 in the absence of the inhibitor, to 3.5 in the presence of 20 mM spermidine. The l‐arginine analog, l‐homoarginine, was also a substrate of the succinyltransferase, and the saturation of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutant growing faster on l‐ornithine than the parent strain had a modified succinyltransferase with a reduced l‐ornithine cooperativity.

The fate of l‐homoarginine was different depending on whether the succinyltransferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalerate was excreted in non‐induced cultures. The ‘waste’ of succinyl CoA, which could not be regenerated from the excreted succinylhomoarginine, explained the inhibition exerted by l‐homoarginine on growth when ornithine or arginine was used as the growth medium.
Induction of the Human Gene for p44, a Hepatitis‐C‐associated Microtubular Aggregate Protein, by Interferon‐α/β
Authors: Kitamura Akiko , Takahashi Kazuhiro , Okajima Ai , Kitamura Naomi
Published: March 2005

A hepatitis‐C‐associated microtubular aggregate protein, referred to as p44, has been identified as a cytoplasmic antigen in the hepatocytes of chimpanzees infected with hepatitis C virus. The production of p44 mRNA is markedly induced in the liver of chimpanzees infected with hepatitis C or hepatitis D virus. To examine the mechanism of this induction, we isolated a genomic clone for the human p44 protein and analyzed its structure. The human p44 gene spans approximately 14 kbp of DNA and consists of nine exons separated by eight introns. An interferon‐stimulated response element, which confers inducibility by interferon‐α/β was found in the promoter region of the gene. Northern‐blot analysis revealed that the human p44 gene is inducible by interferon‐ α/β, but not by interferon‐γ. Functional analysis demonstrated that the interferon‐stimulated response element in the promoter region of the gene mediates the inducibility of the gene by interferon‐α/β. Thus, the human p44 gene is a member of the family of interferon‐α/β inducible genes. The protein p44 may be one of the mediators involved in the antiviral action of interferon.
Relationship between Core Histone Acetylation and Histone H10 Gene Activity
Authors: Girardot Valérie , Rabilloud Thierry , Yoshida Minoru , Beppu Teruhiko , Lawrence Jean‐Jacques , Khochbin Saadi
Published: March 2005

In this study we show a striking correlation between histone H10 gene expression and histone acetylation. Trichostatin A, a highly specific inhibitor of histone deacetylase, efficiently induces H10 gene expression. Moreover, using a cell line sensitive to trichostatin A (FM3A) and a derived cell line selected for its resistance to this inhibitor (TR303), it is shown that the level of H10 gene expression is related to the extent of chromatin acetylation. After showing the S‐phase‐dependent activation of H10 gene expression, we demonstrate that hyperacetylation has a dominant effect on H10 gene expression, since it enhances the expression of the gene independent of the position of cells in the cell cycle. This response to deacetylase inhibitors is specific to H10, since it is not shared by other cell‐cycle‐dependent histone genes (H1 and H4). Finally, by transfection of trichostatin‐A‐resistant and trichostatin‐A‐sensitive cells with a plasmid containing a H10 promoter, we show that the exogenous H10 promoter is also highly sensitive to trichostatin A treatment and that activation of transcription follows exactly the same pattern as activation of the endogenous gene. These data show that histone acetylation may be used to modulate H10 gene activity and offers insight into a possible mechanism in which the developmentally regulated chromatin acetylation acts to potentiate H10 gene expression.
Organisation and Promoter Activity of the Retinoic‐acid‐induced‐heparin‐binding (RIHB) Gene
Authors: Duprez Delphine , Treagger James , Pecqueur Claire , Vigny Marc
Published: March 2005

The heparin‐binding protein, RIHB, is encoded by a gene that is expressed temporarily during the early period of chicken embryogenesis. We have now isolated genomic clones of the chicken RIHB gene, determined its transcription initiation sites, characterized its promoter region and established the functional activity of the RIHB gene promoter. The 5′ flanking region and the beginning of the first exon revealed a structural Organisation characteristic of housekeeping and growth‐control‐related genes. It lacked canonical TATA or CAAT boxes but contained several GC boxes. The structure of the RIHB gene is compared with those of the human and mouse midgestation kidney (MK) genes. In the coding exons, the similarities between the three genes are striking. In contrast, the structure and location of the first non‐coding exon is different. Analysis of the promoter activity suggests an indirect regulation of transcription by retinoic acid and demonstrates a high degree of complexity of the regulation of the RIHB gene.
Crystal Structure of γ‐Oxapentamidine Complexed with d(CGCGAATTCGCG)2
Authors: Nunn Christine M. , Jenkins Terence C. , Neidle Stephen
Published: March 2005

The crystal structure of the complex of γ‐oxapentamidine and the DNA dodecamer d(CGCGAATTCGCG)2 has been determined to a resolution of 0.22 nm and an R factor of 18.9%. The γ‐oxapentamidine ligand interacts with the dodecamer by classic minor groove binding via interactions within the Arich region of the minor groove. A chain of solvent molecules lies along the mouth of the minor groove on the outside of the bound ligand. The structural details of the complex are discussed and compared with the closely analogous complex with pentamidine bound to the same dodecamer [Edwards, K. J., Jenkins, T. C. & Neidle, S. (1992) Biochemistry 31, 7104–7109]. The amidinium groups of the ligand do not hydrogen bond to bases, but are in close contact with O4′ sugar ring atoms. This in part explains the reduced DNA binding affinity of this ligand compared to pentamidine.
Interaction of Tryptophan Residues of Cytochrome P450scc with a Highly Specific Fluorescence Quencher, a Substrate Analogue, Compared to Acrylamide and Iodide
Authors: Lange Reinhard , Anzenbacher Pavel , Müller Sylvaine , Maurin Luc , Balny Claude
Published: March 2005

The cytochrome P450scc tryptophan fluorescence was studied by the use of the three quenchers acrylamide, 25‐doxyl‐27‐nor‐cholesterol (CNO) and potassium iodide (KI). All the nine tryptophan residues were accessible to acrylamide. Whereas a strong interacation (static quenching) between acrylamide and tryptophan in the active site had been found previously for cytochrome P450c21 [Narasimhulu, S. (1988) Biochemistry 27, 1147–1153], in the case of P450scc the temperature dependence of the slope of the linear Stern‐Volmer plots indicated a dynamic quenching mechanism. This mechanism was confirmed by fluorescence lifetime measurements. Of the three observed lifetimes τ1= 3.1 ± 0.5 ns, τ2= 0.7 ± 0.25 ns and τ3= 20 ± 10 ps, τ1 decreased noticably as a function of the acrylamide concentration. CNO, a spin‐labeled substrate which is known to bind tightly to the substrate‐binding site of P450scc, quenched 15.5% of the total fluorescence. The Lehrer plot of this compound indicated a static quenching process with a reciprocal quenching constant of 1/Ks= 4 μM, a value which is in accord with the dissociation constant. Our data indicate that CNO quenches selectively one or two tryptophan residue(s) in the acive site. The fluorescence spectrum of the residue(s) accessible to CNO was characterized by a red‐shifted emission maximum (from 332 nm to 336 nm). The same residue(s) appeared to be quenched by potassium iodide, although much less effectively (1/Ks= 0.12 M). The most probable candidate for a complex formation with CNO is Trp417, which is rather close to Cys422 (the fifth heme ligand). Four arginine residues (Arg411, Arg420, Arg425 and Arg426) in the heme peptide may constitute the iodide‐binding site.