bluuebunny/crossref_metadata_2025 huggingface dataset:
Trimethylphosphine binding to horse‐heart and sperm‐whale myoglobins
Authors: BRUNEL Christian , BONDON Arnaud , SIMONNEAUX Gérard
Published: March 2005

Two‐dimensional nuclear magnetic resonance techniques have been used to assign resonances corresponding to the heme pocket and several other residues of horse heart and sperm whale myoglobins ligated by trimethylphosphine. The assignment procedure was based mainly on the nuclear Overhauser effect connectivities with the ligand and the heme substituents. For quantitative measurements of Overhauser effects, application of truncated driven techniques between a proton from distal residues and methyl groups from the ligand was used to determine internuclear distances. These new results have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between horse heart and sperm whale myoglobins. The interproton distances between distal amino acid residues and trimethylphosphine were found to be longer in horse heart myoglobin relative to those in sperm whale myoglobin. This result suggests that the size of the heme pocket is larger in horse heart myoglobin.

Association and dissociation rate constants were measured for trimethylphosphine binding to myoglobins. Both values were four times larger for horse heart myoglobin than those for sperm whale myoglobin. This observation confirms the structural results obtained with NMR studies and is rationalized by a greater stabilization of a larger pocket in horse heart myoglobin relative to sperm whale myoglobin.
Determination of the sequence requirements for the expression of a Xenopus borealis embryonic/larval skeletal actin gene
Authors: LAKIN Nicholas D. , BOARDMAN Mark , WOODLAND Hugh R.
Published: March 2005

In this study, we demonstrate that all sequences necessary and sufficient for the expression of a Xenopus borealisα3B embryonic/Iarval skeletal actin gene, reside in a 156‐nucleotide fragment of the promoter that spans nucleotides ‐197 to ‐42. This region of the promoter contains three imperfect repeats of the CC(A/T)6GG (CArG) box motif that have been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion of the actin promoter, using Xenopus microinjection techniques as a transient assay system for promoter activity, shows that the most distal CArG box (CArG box3) is essential for the full expression of the gene. Under our assay conditions, the most proximal CArG box (CArG box1) exhibits two binding activities using band‐shift analysis. One of these binding activities contains components antigenically related to a serumresponse factor (transcription factor), whilst the second does not. In contrast, CArG box3 produces only a single retarded band using electrophoretic mobility‐shift analysis. Although the shifted complex coelectrophoreses with the CArG box1/serum‐response factor complex, the band produced by CArG box3 appears to be distinct from SRF. In addition to the CArG motifs, a further upstream regulatory element has been identified in the actin promoter between nucleotides ‐197 and ‐167. In the actin promoter, a downstream region can apparently fulfil this function.
The addition of nine residues at the C‐terminus of human prolactin drastically alters its biological properties
Authors: GOFFIN Vincent , STRUMAN Ingrid , GOORMAGHTIGH Erik , MARTIAL Joseph A.
Published: March 2005

We have added nine extra residues to the C‐terminal of human prolactin and analysed the effect of this mutation on the ability of the hormone to bind to its lactogenic receptor and to induce Nb2 cell division. Both properties are markedly affected when compared to the natural 23‐kDa human prolactin. Since no alteration of the global protein folding was detected either by circular dichroism or by infrared spectroscopy, the decrease in biological potency can be exclusively attributed to an effect of the nine additional residues on their near environment. From infrared analysis and secondary structure prediction, the elongated tail is assumed to be involved in a β‐sheet with a few residues initially belonging to the fourth helix. Moreover, from the X‐ray structures of porcine and human growth hormones, two proteins homologous to prolactins, the nine extra residues are likely to fold within a concave pocket delimited by helices 1 and 4, and the second half of the loop connecting helices 1 and 2 (loop 1). Thereby, we suggest that the additional residues prevent some residues belonging to this pocket from interacting with the lactogenic receptor. This is in perfect agreement with our earlier proposal that the binding site of prolactin to the lactogenic receptor is homologous to that of growth hormone to the somatogenic receptor, i.e. essentially composed of residues belonging to this concave pocket.
Interaction of calmodulin with a putative calmodulin‐binding domain of inositol 1,4,5‐triphosphate 3‐kinase.
Authors: ERNEUX Christophe , MOREAU Colette , VANDERMEERS André , TAKAZAWA Kazunaga
Published: March 2005

Recombinant rat brain inositol 1,4,5‐triphosphate [Ins(1,4,5)P3] 3‐kinase was expressed in Escherichia coli as a β‐galactosidase fusion product. It could be adsorbed onto calmodulin‐Sepharose and eluted in Ca2+‐free medium as a 48‐kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin‐binding domain on Ins(1,4,5)P3 3‐kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin‐binding domain. The inhibition was mimicked by a well‐characterized peptide derived from the sequence of smooth muscle myosin light‐chain kinase calmodulin‐binding site. (b) The construction of two mutants by site‐directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.
A novel cAMP‐dependent regulatory region including a sequence like the cAMP‐responsive element, far upstream of the human CYP21A2 gene
Authors: WATANABE Nobuaki , KITAZUME Masaru , FUJISAWA Jun‐ichi , YOSHIDA Mitsuaki , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

Deletion mutants in the 5′ upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP‐dependent expression of the gene in the transient expression system using Y‐1 cells (mouse adrenocortical tumor cell line). This regulatory element (‐2574 to ‐2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP‐responsive element (CRE) and a CAAT‐like sequence. The CRE‐like sequence has a five‐base motif (5′‐TGACG‐3′) of the palindromic CRE consensus (TGACGTCA).

Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT‐like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue‐specific enhancers (adrenal‐specific protein factor and Ad4‐like sequences) and their binding factors to express a high level of cAMP‐responsive expression of the CYP21A2 gene.
Amino acid sequence of an intracellular, phosphate‐starvation‐induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells
Authors: LÖFFLER Andreas , GLUND Konrad , IRIE Masachika
Published: March 2005

The primary structure of an intracellular ribonuclease (RNase LX) from cultured tomato (Lycopersicon esculentum) cells has been determined. Previous studies have shown that the protein is located inside the tomato cells but outside the vacuoles and that its synthesis is induced after depleting the cells for phosphate [Löffler, A., Abel, S., Jost, W., Beintema, J. J., Glund, K. (1992) Plant Physiol. 98, 1472–1478]. Sequence analysis was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the protein. RNase LX consists of 213 amino acids and has a molecular mass of 24300 Da and an isoelectric point of 5.33. The enzyme contains 10 half‐cystines and there are no potential N‐glycosylation sites detectable in the sequence. RNase LX, as compared to an extracellular tomato RNase (RNase LE), which is also phosphate regulated and the amino acid sequence of which was recently established [Jost, W., Bak, H., Glund, K., Terpstra, P. & Beintema, J. J. (1991) Eur. J. Biochem. 198, 1–6] has 60% of all amino acids identical and in identical positions, revealing a high degree of similarity between both proteins. In contrast to RNase LE, RNase LX has a C‐terminal extension of nine amino acids. The C‐terminal tetrapeptide HDEF may be a retention signal of the protein in the endoplasmic reticulum.
The role of Cys12, Cys17 and Arg18 in the catalytic mechanism of low‐Mr cytosolic phosphotyrosine protein phosphatase
Authors: CIRRI Paolo , CHIARUGI Paola , CAMICI Guido , MANAO Giampaolo , RAUGEI Giovanni , CAPPUGI Gianni , RAMPONI Giampietro
Published: March 2005

Low‐Mr phosphotyrosine protein phosphatase (PTPase), previously known as low‐Mr acid phosphatase, catalyzes the in‐vitro hydrolysis of tyrosine phosphorylated proteins, low‐Mr aryl phosphates and natural and synthetic acyl phosphates. Its activity on Ser/Thr‐phosphorylated proteins and on most alkyl phosphates is very poor. In this study the mechanism of benzoyl‐phosphate hydrolysis was studied by means of non‐mutated and mutated PTPase fusion proteins. The mechanism of benzoyl‐phosphate hydrolysis catalyzed by the enzyme was compared to the known mechanism of p‐nitrophenyl‐phosphate hydrolysis. The results demonstrated that both hydrolytic processes proceed through common enzyme‐catalyzed mechanisms. Nevertheless, the performed phospho‐enzyme‐trapping experiments enable us to identify Cys12 as the active‐site residue that performs the nucleophilic attack at the phosphorus atom of the substrate to produce a phosphoenzyme covalent intermediate. In addition, while the role of Cys17 in the substrate binding was confirmed, its participation a second time in the step that involves the Cys12 dephosphorylation was suggested by the results of phosphoenzyme‐trapping experiments. The participation of Arg18 in the substrate‐binding site was demonstrated by site‐directed mutagenesis that produced the conservative Lys18 and the non‐conservative Met18 mutants. Both these mutants were almost inactive and not able to bind the substrate and a competitive inhibitor. Furthermore, phosphoenzyme‐trapping experiments clearly excluded that Cys62 and Cys145 (that were indicated by another laboratory to be involved in the active site of the enzyme as powerful nucleophilic agents) are the residues directly involved in the formation of the phosphoenzyme covalent intermediate.
Polyamine‐mediated regulation of S‐adenosylmethionine decarboxylase expression in mammalian cells
Authors: STJERNBORG Louise , HEBY Olle , MAMONT Pierre , PERSSON Lo
Published: March 2005

Cell proliferation is dependent on an adequate supply of the polyamines putrescine, spermidine and spermine. One of the key steps in the polyamine biosynthetic pathway is catalyzed by S‐adenosylmethionine decarboxylase (AdoMetDC). In the present study we have used a newly synthesized enzyme‐activated irreversible AdoMetDC inhibitor, 5′‐{[(Z)‐4‐amino‐2‐butenyl]methyl‐amino}‐5′‐deoxyadenosine [(Z)‐AbeAdo], to investigate the regulation of this enzyme. Treatment of mouse L1210 leukemia cells with (Z)‐AbeAdo resulted in a total inhibition of their AdoMetDC activity followed by depletion of the spermidine and spermine content. The putrescine content, however, was dramatically increased after treatment with (Z)‐AbeAdo. In spite of the cellular depletion of spermidine and spermine, only a minor inhibitory effect was obtained on cell growth, indicating that putrescine at a high concentration might partly replace spermidine and spermine in their growth‐promoting functions. Cells grown in the presence of (Z)‐AbeAdo exhibited an increased synthesis of AdoMetDC, which was counteracted by the addition of either spermidine or spermine. The change in AdoMetDC synthesis could not be fully explained by a change in the level of AdoMetDC mRNA, indicating also a translational control. Mammalian AdoMetDC is synthesized as a larger proenzyme, which is then cleaved into two subunits of different sizes. The conversion of the proenzyme into the subunits is a very rapid process, which is stimulated greatly by putrescine in vitro. However, the processing of the proenzyme in the (Z)‐AbeAdo‐treated L1210 cells was not affected by their very high putrescine content, indicating that the conversion might be saturated at low levels of putrescine, or that most of the putrescine in the (Z)‐AbeAdo‐treated L1210 cells might be bound to sites normally occupied by spermidine and spermine.
O‐Linked neutral sugar chains of porcine zona pellucida glycoproteins
Authors: HIRANO Toshio , TAKASAKI Seiichi , HEDRICK Jerry L. , WARDRIP Nate J. , AMANO Junko , KOBATA Akira
Published: March 2005

The O‐linked chains were liberated from porcine zona pellucida glycoproteins by alkaline‐borohydride treatment and the structures of neutral oligosaccharides were determined. The major oligosaccharides had the following structures with type‐1 cores, (Galβ1→4GlcNAcβ1→3)t‐3‐Galβ1→3N‐acetylgalactosaminitol (54%) and GlcNAcβ1→3(Galβ1→4GlcNAcβ1→3)0–1Galβ1→3N‐acetyl‐galactosaminitol (16%). Approximately 6% of the oligosaccharides had the structure with a type‐3 core, Galβ1→4GlcNAcβ1→3N‐acetylgalactosaminitol. Oligosaccharides having α‐galactose (2%) and β‐GalNAc (4%) at their non‐reducing termini were also found as minor components.
Effects of light stress on the expression of early light‐inducible proteins in barley
Authors: PÖTTER Eyck , KLOPPSTECH Klaus
Published: March 2005

Treatment of six‐day‐old barley leaves with white light of high intensity, 250–2000 W/m2, leads to a linear increase in the steady‐state concentrations of early light‐inducible protein (ELIP) mRNA followed by an accumulation of the protein. Accumulation of ELIP mRNA, under light stress, is highest in the basal third of the leaf and declines to approximately 50% of this level in the apical segment. The amount of the accumulated protein decreases more steeply towards the tip than would be expected from mRNA levels. This finding, as well as the fact that during greening a massive accumulation of the protein starts only at a time when the steady‐state concentrations of ELIP mRNA have declined to 10% of the maximal value, indicate post‐transcriptional control. Accumulation is presumably achieved by stabilization of the protein. ELIP mRNA and protein levels, induced by a 2‐h period of high‐light stress, are lowest in the afternoon and highest at midnight and during the morning. The inducibility of ELIP by high light is therefore under diurnal control. An increase of light stress, due to application of the carotenoid‐biosynthesis inhibitor norfluorazon, results in a considerable induction of ELIP mRNA and protein. The plant hormone abscisic acid exerts only a small effect on the mRNA level. In all cases studied, the light‐induced increase in the amount of ELIP mRNA was accompanied by a corresponding decline in the mRNA levels for the apoprotein of the chlorophyll‐alb‐binding protein. Steady‐state concentrations of mRNA for the small subunit of ribulose‐1,5‐bisphosphate carboxylase were hardly affected under all investigated light intensities.
Tissue‐specific molecular diversity of amidating enzymes (peptidylglycine α‐hydroxylating monooxygenase and peptidylhydroxyglycine N‐C lyase) in Xenopus laevis
Authors: IWASAKI Yasuno , SHIMOI Hiroko , SAIKI Hiromasa , NISHIKAWA Yoshiki
Published: March 2005

We investigated the molecular diversity of the paired enzymes, peptidylglycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase (PHL), involved in peptide C‐terminal amidation. Three kinds of amidating enzyme (AE) cDNAs (AE‐I, AE‐II and AE‐III) have previously been isolated from Xenopus laevis skin. While AE‐I cDNA encodes only PHM, AE‐III cDNA encodes a protein containing both PHM and PHL sequences and a transmembrane domain. On the other hand, the translated product of AE‐II has not been detected yet. Endoproteolytic cleavage of the AE‐III protein generates separated forms of PHM and PHL that are purified from X. laevis skin. Expression of AE‐III in insect cells using a baculovirus expression vector system indicated that PHM and PHL exist as a membrane‐associated, bifunctional enzyme without endoproteolysis in insect cells.

Both PHM and PHL activities were detected in all the X. laevis tissues examined. Particularly, the highest levels of both activities were found in skin, brain and heart. We identified basically three types of enzymes in X. laevis; soluble PHM, soluble PHL and a membrane‐associated, bifunctional enzyme that has both PHM and PHL domains. While the skin contained soluble types of PHM and PHL, the brain and heart predominantly contained the membrane‐associated, bifunctional type. Analysis of mRNA levels by the reverse‐transcript polymerase chain reaction method and Western blot analysis using PHM‐specific antibody revealed that such molecular diversity of PHM and PHL among the tissues are produced by changing the ratio of AE‐I mRNA/AE‐III mRNA, and by endoproteolytic processing of the membrane associated precursor protein.
The conformation of new tetrahydropyrimidine derivatives in solution and in the crystal
Authors: INBAR Livia , FROLOW Felix , LAPIDOT Aviva
Published: March 2005

We have recently identified by NMR techniques two new tetrahydropyrimidine derivatives, 2‐methyl‐4‐carboxy‐5‐hydroxy‐3,4,5,6‐tetrahydropyrimidine [THP(A)] and 2‐methyl‐4‐carboxy‐3,4,5,6‐tetrahydropyrimidine [THP(B)], which accumulated in actinomycin D producing Streptomyces parvulus. Their relatively high intracellular concentrations during actinomycin D synthesis and identity of the time of onset of their synthesis with that of actinomycin D synthesis leads us to suggest that they may function in the self‐defense mechanism of actinomycin‐producing organisms. Here we present a combination of one‐dimensional and two‐dimensional 1H‐ and 13C‐NMR studies in solution and X‐ray crystallography of THP(A) and THP(B). Our results demonstrate identical conformations of THP(A) and of THP(B) in H2O solution and in the crystal. THP(A) and THP(B) form zwitterionic molecules with the half‐chair conformation. Both techniques, NMR and X‐ray crystallography, indicate that the carboxyl group of THP(B) is in an axial position and that the carboxyl and the hydroxyl groups of THP(A) are also in axial positions. The coplanar zigzag configurations (H4 C4 C5 C6 H6) of THP(A) and THP(B), deduced from NMR coupling measurements, were confirmed by X‐ray diffraction studies. The crystal structure of THP(B) indicates that the hydrogen atoms of the methyl group are disordered, exhibiting two distinct conformations, whereas the methyl group hydrogens of THP(A) exhibit only one conformation. This finding is in accordance with the dynamic properties of THP(A) and THP(B) derived from carbon spin‐lattice relaxation rate measurements. In spite of a different crystal packing and considerable differences in the dynamic properties (Ti) in solution, the two molecules exhibit an almost identical conformation. The similar conformation of the two different crystals can be considered as evidence for a relatively high internal stability of the molecule against intermolecular forces.
Molecular cloning and functional characterization of V2 [8‐lysine] vasopressin and oxytocin receptors from a pig kidney cell line
Authors: GORBULEV Valentin , BÜCHNER Hubert , AKHUNDOVA Aida , FAHRENHOLZ Falk
Published: March 2005

[Arg8]vasopressin and oxytocin are the two main members of the neurohypophysial hormone family found to be present in nearly all mammals. [Lys8]vasopressin ([Lys8]VP) has been identified as the antidiuretic hormone in pig and some marsupial families. The porcine‐derived kidney epithelial cell line, LLC‐PK1, expresses both [Lys8]VP receptors coupled to the activation of adenylate cyclase (V2 receptors) and oxytocin receptors. Here we report the molecular cloning of the V2 [Lys8]VP receptor and the oxytocin receptor from LLC‐PK1 cells. The cloned V2 [Lys8]VP receptor differs from human and rat V2 [Arg8] receptors mainly in its N‐terminal region, in residues located in the extracellular loops and in intracellular phosphorylation sites. When expressed in COS7 cells, the V2 [Lys8]VP receptor exhibits the relative order of ligand affinity [Lys8]VP = [Arg8]VP ≫ 1‐deamino[d‐Arg8]VP ≥ oxytocin and adenylate‐cyclase stimulation, expected for the porcine V2 [Lys8]VP receptor but different from V2 [Arg8]VP receptors. Adenylate‐cyclase activation by [Lys8]VP was inhibited in COS7 cells by a V2 antagonist. The cloned oxytocin receptor exhibits in COS7 cells a ligand specificity typical of mammalian oxytocin receptors. mRNA‐distribution analysis revealed a single 5.5‐kb transcript in the uterus from pregnant guinea pig.
A study of the binding of NADP coenzymes to dihydrofolate reductase by raman difference spectroscopy
Authors: ZHENG Jie , CHEN Yong Q. , CALLENDER Robert
Published: March 2005

We report here the Raman spectra of NADPH, NADP+, 3‐acetylpyridine adenine dinucleotide (AcPdADP+), NADH and a fragment of these molecules, 2′‐phospho‐adenosine‐5′‐diphosphoribose (Ado2′p5′ppRib), bound to Escherichia coli dihydrofolate reductase (DHFR). The positions that are observed for the bound adenosine ‘triplet’ bands are consistent with a protein binding pocket for this group which is quite hydrophobic in nature. No binding effect is observed on Raman bands associated with the nicotinamide group of NADP+ as a binary complex with DHFR, suggesting very loose, if any, binding of this group. In contrast, changes in the Raman spectrum of the nicotinamide group of NADP+ bound to an inhibitor (trimethoprim) ternary complex of DHFR are clearly observed which indicate substantial binding interaction. The carboxamide group of bound NADPH (and NADH) adopts the trans conformation. A 35‐cm−1 upshift is observed in the rocking motion of the carboxamide ‐NH2 group of NADPH, and a 5‐cm−1 upward shift is seen in the C = O stretch mode of AcPdADP+ upon binding to the enzyme‐trimethoprim complex. These results suggest that the –NH2 group of the carboxamide moiety is more tightly hydrogen bonded in the protein binding pocket than in solution while that of the C = O group is less tightly hydrogen bonded; these hydrogen bonds would appear to be responsible for holding the nicotinamide headgroup in place properly for catalysis. We have compared this with the results obtained previously in other protein complexes, and interpret the observed shifts in these bands as a measure of the hydrogen bonding enthalpy of the –NH2 and C = O groups with their protein environments. Perhaps surprisingly, the magnitude of the hydrogen bonding enthalpy takes on a limited number of discrete values over five protein complexes rather than over a continuous range. The effect that this has on the catalytic properties of DHFR and the other NAD dehydrogenases that we have studied to date, particularly their stereo‐chemistry, is discussed. A small downward shift is observed for the P O stretch of the 2′‐phosphate moiety of NADP. This indicates that the 2′‐phosphate moiety binds to DHFR in the dianionic form. Furthermore, the local enthalpic interaction that the 2′‐phosphate group has with protein is stronger than its interaction with water.
Detection of the in vivo incorporation of a metal cluster into a protein
Authors: GOLLAN Ute , SCHNEIDER Klaus , MÜLLER Achim , SCHÜDDEKOPF Kerstin , KLIPP Werner
Published: March 2005

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo‐independent nitrogen‐fixing system, an ‘iron‐only’ nitrogenase which is strongly repressed by molybdate. The MoO2− concentration causing 50% repression of the alternative nitrogenase in nifHDK− cells was 6 nM. If MoO42− was added to a growing nifHDK− culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H6 formation decreased continously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yeild of C2H6 increased from 2% (C2H6/C2H4X100) in the absence of MoO42−, to a maximal value of 69% in the presence of MoO42− (1 mM) after 72 h incubation. This ‘Mo effect’ appeared to be stronger the higher the MoO42− concentration in the medium and the longer the incubation time. In the presence of ReO4−, WO42− or VO43−, a similar effect did not occur.

The ‘Mo effect’ was not observed in a nifHDK−nifE− double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK−nifQ− mutant.

Crude extracts from nifHDK− cells cultivated in the presence of MoO42−, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S= 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the ‘Mo effect’ is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase.

The ‘Mo effect’ was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the ‘Mo effect’ appeared, therefore, to be independent of de‐novo protein synthesis. The analysis of nifE‐lacZ and nifN‐lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO42− deficiency.

The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.
A high‐order structure of plant storage proprotein allows its second conversion by an asparagine‐specific cysteine protease, a novel proteolytic enzyme
Authors: MURAMATSU Masayoshi , FUKAZAWA Chikafusa
Published: March 2005

During seed embryogenesis, glycinin, the 11‐S seed storage protein found in soybeans, undergoes post‐translational proteolytic processing, in which a proprotein molecule is cleaved into an acidic and a basic subunit by a one‐point cleavage that occurs at the carboxyl side of the asparaginyl residue located at the junction of the subunits. To elucidate the mechanism of this very limited proteolysis, we purified the cysteine endoprotease and used purified proglycinin produced by Escherichia coli as a substrate. This enzyme was separated by isoelectric focusing into three isomeric forms: two had a molecular mass of 33 kDa and the third, 33.8 kDa. The cysteine protease was found both in the proteinaceous vacuoles of cotyledonary tissue of immature seeds and in mature seeds, and is the first proteolytic enzyme to be classified as an asparagine‐specific endoprotease. The results also indicate that the above proteolysis is largely attributable to the conformational accessibility of the enzyme to the asparaginyl residue in the cleavage site of proglycinin. The conformation of this single enzyme‐accessible region on the proglycinin molecule is relatively flexible and becomes unstable under low salt conditions, or when heat is applied, causing the enzyme to lose its specificity.
Late‐fibrin(ogen) fragment E modulates human α‐thrombin specificity
Authors: BOUTON Marie‐Christine , JANDROT‐PERRUS Martine , BEZEAUD Annie , GUILLIN Marie‐Claude
Published: March 2005

Fibrinogen contains at least two independent sites having demonstrable affinity for α‐thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion‐binding exosite of α‐thrombin. Taking advantage of its solubility, we have used late‐fibrin(ogen) fragment E in competition experiments to examine its effect on α‐thrombin specificity. We show that fragment E modulates α‐thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin‐thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of α‐thrombin‐catalyzed fibrinopeptide‐A cleavage (Ki= 5.2±1.3 μM), indicating that α‐thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited α‐thrombin‐induced serotonin release by platelets (concentration required to obtain 50% inhibition IC50= 10 μM) and α‐thrombin binding to GPIb. Fragment E competitively inhibited α‐thrombin binding to thrombomodulin (Ki= 18.3±0.8 μM) but did not inhibit protein‐C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non‐identical sites, while protein C binds to an independent site on α‐thrombin.
Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus‐infected Sf21 cells
Authors: GRABENHORST Eckart , HOFER Bernd , NIMTZ Manfred , JÄGER Volker , CONRADT Harald S.
Published: March 2005

Human interleukin 2 (IL‐2) and human IL‐2 mutant proteins, with artificially introduced N‐glycosylation or O‐glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL‐2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N‐terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3–5 amino acids were also absent in a small proportion of the purified proteins.

The introduction of potential artificial O‐glycosylation peptide sequences (¨GGKAPTPPPK¨), to the C‐terminus or between positions 80 and 81 of the IL‐2 polypeptide chain, resulted in the secretion of unglycosylated and O‐glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(β1–3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL‐2 mutant protein. The Gal(β1–3)GalNAc protein forms could be partially altered in vitro to mammalian‐type glycoforms by porcine liver β‐galactoside α‐2,3‐sialyltransferase in the presence of CMP‐N‐acetylneuraminic acid.

An IL‐2 mutant form, with an 11‐amino‐acid peptide of human interferon‐β at position 4, which includes its only N‐glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(α1–6)]GlcNAc or Man2GlcNAc[Fuc(α1–6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell‐culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.
Lipase from Pseudomonas aeruginosa
Authors: OSHIMA‐HIRAYAMA Noriko , YOSHIKAWA Kazuhiro , NISHIOKA Takaaki , ODA Jun'ichi
Published: March 2005

The lipase gene from Pseudomonas aeruginosa TE3285 is followed by another gene, lipB. The lipase gene was expressed in Escherichia coli BL21(DE3)pLysS using the T7 RNA polymerase expression system. The mature lipase was accumulated as inclusion bodies at 42% of the total cell proteins. The inclusion bodies were solubilized with 8 M urea, but lipase activity was not detected in the solubilized preparation containing 85% lipase protein even after removing urea by dialysis. The lipB gene, positioned downstream of the lipase gene and thought to be necessary for the expression of the lipase gene, was expressed in Escherichia coli JM109 as a fusion with the glutathi‐one transferase gene from Schistosoma japonicum. The fusion protein was partially purified on glutathione–agarose beads to 36% purity. Incubated with the fusion protein at a molar ratio of 1:1 at 4°C for 24 h, the solùbilized lipase showed lipase activity of about a tenth that of the purified lipase prepared from Pseudomonas aeruginosa TE3285. Magnesium ions and ATP were not essential but increased the activation. When the fusion protein was treated with thrombin to release the glutathione transferase part, it retained its activity. The lipase activation with lipB protein probably proceeds to form a 1:1 complex with the inactive, solubilized lipase protein but by a different mode from known chaperones.
Overproduction of S‐adenosylmethionine decarboxylase in ethylglyoxal‐bis(guanylhydrazone)‐resistant mouse FM3A cells
Authors: SUZUKI Toshikazu , SADAKAT Yasuyo , KASHIWAGI Keiko , HOSHINO Kenji , KAKINUMA Yoshimi , SHIRAHATA Akira , IGARASHI Kazuei
Published: March 2005

A variant cell line, termed SAM‐1, which overproduced S‐adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five‐times higher than control cells. The rate of AdoMetDC syntheis and the amount of AdoMetDC existing in SAM‐1 cells were about five‐times those in control cells. The amount of AdoMetDC mRNA existing in SAM‐1 cells was five‐times more than that in control cells. The amount of 5′‐([(Z)‐4‐amino‐2‐butenyl]methylamino]‐5′‐deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five‐times more in SAM‐1 cells than in control cells. However, the following were the same in both SAM‐1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5′‐untranslated region of AdoMetDC mRAN, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2= about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM‐1 cells. The results indicate that the overproduction of AdoMetDC in SAM‐1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regultion of AdoMetDC and the physiological function of polyamines.
Monoclonal antibodies against brain acetycholinesterases which recognize the subunits bearing the hydrophobic anchor
Authors: LIAO Jian , MORTENSEN Vibeke , NØRGAARD‐PEDERSEN Bent , KOCH Claus , BRODBECK Urs
Published: March 2005

Monoclonal antibodies were raised against amphiphilic detergent‐soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132‐4 (IgG), 132‐5 (IgG), and 132‐6 (IgG3), specific for brain DS‐AChE were selected and subcloned. These mAb reacted with native as well as heat‐denatured and SDS‐denatured DS‐AChE, indicating that the epitopes to which mAb bounds are continuous determinants. The mAb cross‐reached with DS‐AChE from bovine and mouse brain and with brain DS‐AChE from river trout (Salmo trutta forma fario) and lake trout (Salmo trutta forma lacustria). No cross‐reaction was detected with the following antigens: salt‐soluble (SS) AChE from bovine brain, glycophospholipid‐anchored AChE from human and bovine erythrocytes, DS‐butrycholinesterase and SS‐butyrylcolinesterase (BtChE) from the brains of human and bovine, DS‐BiChE from chicken and BiChE from human serum. Deglycosylation of brain DS‐AChE with N‐glycosidase F did not abolish the binding of mAb to DS‐AChE. After reduction of brain DS‐AChE by dithiothereitol, the mAb on longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS‐AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS‐AChE. Sucrose‐density‐gradient centrifugation showed the mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western bolt, after SDS/PAGE under non‐reducing conditions, showed the mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, trimers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132‐4, 132‐5 and 132‐6 recognized DS‐AChE form fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.
The bis(adenosin‐N6‐yl) alkanes, a family of potential dinucleoside polyphosphate analogue precursors
Authors: CHEN Haijuan , McLENNAN Alexander G.
Published: March 2005

The potential diadenosine polyphosphate analogue precursor, bis(adenosin‐N6‐yl)dodecane (A[CH2]12A) (Chen, H. & McLennan, A. G. (1993) Eur. J. Biochem. 213, 935–944.) is equally toxic to both wild‐type and adenosine‐kinase‐deficient BHK cells at concentrations up to 100 μM; at higher concentrations, wild‐type cells are more sensitive, as are cells over‐expressing adenosine kinase. Thus both the nucleoside and its nucleotide products are toxic. In contrast to adenosine toxicity, the toxicity of A[CH2]12A to S‐49 T‐lymphoma cells could not be reversed by uridine or by l‐homocysteine thiolactone. A[CH2]12A and all its shorter chain bis(adenosin‐N6‐yl)alkane homologues could relieve the toxicity of low adenosine concentrations (<20 μM) to S‐49 cells, mainly through inhibition of adenosine kinase, while relief of the toxicity of high adenosine concentrations (>20 μM) required the longer chain homologues. A[CH2]12A at 10 μM completely eliminated adenosine toxicity. Deoxyadenosine toxicity could also be relieved, but only that due to low concentrations (<4 μM). A[CH2]12A had only a slight stimulatory effect on S‐adenosylhomocysteine‐hydrolase activity.
Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP
Authors: LÖW Andreas , SPRINZL Mathias , FAULHAMMER Heinz G.
Published: March 2005

The amino acid sequence motifs of human c‐H‐ras p21 involved in the interaction with guanosine nucleotides were cross‐linked to in situ periodate‐oxidized [α‐32P]GDP or [α‐32P]GTP. Sitespecific reaction was achieved by cross‐linking conserved lysine residues close to the G‐nucleotide binding site of p21 with the 2′,3′ ‐dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp‐N digestion, HPLC separation of 32P‐labeled peptides and N‐terminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N‐K‐X‐D and S‐A‐K/L consensus elements of ras proteins, respectively, were identified. No significant divergences in the position and extent of covalent modification could be detected between p21 · GDP and p21 · GTP. This is in contrast to Thermus thermophilus EF‐Tu · GDP and EF‐Tu · GTP, which were investigated with the same technique [Peter, M. E., Wittmann‐Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132–9139] and which exhibited considerable differences in cross‐linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross‐linking [α‐32P]GTP with GTP‐binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.
Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1‐ATPase β subunit
Authors: LOPEZ‐MEDIAVILLA Casilda , VIGNY Hélène , GODINOT Catherine
Published: March 2005

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1‐induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron‐transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity.

IF1 antibodies have been used to show that IF1 can bind not only to the β subunit of F1‐ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339–1344] but also to a protein present in the inner‐mitochondrial membrane. The cross‐linking of IF1 to this membrane protein gave a product of Mr 15000–16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross‐linked product was obtained by using a cleavable cross‐linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent Mr 5000–6000.

The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross‐linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X‐100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory‐binding site at the β subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate Mr 5000–6000.
The glucocorticoid receptor acts as an antirepressor in receptor‐dependent in vitro transcription
Authors: ERIKSSON Per , WRANGE Örjan
Published: March 2005

Glucocorticoid‐receptor‐dependent and glucocorticoid‐response‐element‐dependent in vitro transcription was established using a crude nuclear extract and purified glucocorticoid receptor from rat liver. The capacity of glucocorticoid receptor to stimulate in vitro transcription was only detectable when basal transcription, i.e. transcription in the absence of glucocorticoid receptor, had been repressed. Transcriptional repression was achieved either by adding purified histone H1, or by lowering the amount of DNA template relative to the amount of crude nuclear extract. Glucocorticoid receptor caused a 1.1±0.7‐fold stimulation of transcription from the mouse‐mammary‐tumorvirus promoter when basal transcription was not repressed, and a 7.0±1.5‐fold stimulation when basal transcription had been repressed by addition of histone H1. Similar results were obtained when using a minimal promoter consisting of two glucocorticoid‐response elements and a TATA box. Our data suggest that glucocorticoid receptor stimulates in vitro transcription by an antirepression mechanism.
Site specificity of glycation of horse liver alcohol dehydrogenase in vitro
Authors: SHILTON Brian H. , CAMPBELL Robert L. , WALTON Donald J.
Published: March 2005

The site specificity of in vitro glycation of horse liver alcohol dehydrogenase (ADH) was examined and the results interpreted in terms of structural features of the enzyme molecule. In a phosphate buffer solution, glycation occurred at Lys231 (the main site of glycation in vivo), at Lys228 (which is not glycated in vivo), and at several unidentified positions. Buffer anions or NAD+ did not affect glycation of Lys231; this supported our hypothesis that the base catalyst which removes a proton from carbon 2 of a Lys231‐attached aldimine is part of the ADH molecule [Shilton, B. H. & Walton, D. J. (1991) J. Biol. Chem. 266, 5587–5592]. Use of a molecular modelling programme indicated that this catalyst was likely to be the imidazole group of His348, exerting its effect through the hydroxyl of Thr347. Glycation of Lys228 occurred only in the presence of phosphate; in this case molecular modelling showed that the base catalyst could be a phosphate ion, bound to ADH at a positive region of the coenzyme binding site. NAD+ inhibited glycation of Lys228 by binding to the enzyme and restricting access to glucose.
Estrogen‐inducible pS2 protein is not the key regulatory component in the proliferation of human breast cancer cells (MCF‐7)
Authors: KIDA Noriyoshi , YOSHIMURA Tomoaki , TAKAHASHI Haruo , NAGAO Seiji , NOZAWA Yoshinori , FURUKAWA Yoshiko , MORI Kazutoshi , HAYASHI Kyozo
Published: March 2005

Exposure of human breast cancer cells (MCF‐7) to tumor promoters such as 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) for 24 h at concentrations of 1–100 nM resulted in marked inhibition of DNA synthesis but a 3–5‐fold increase in the amount of pS2 protein in the medium. These results support our previous suggestion that pS2 protein is not involved in the mechanism controlling proliferation of MCF‐7 cells. During treatment with TPA, the intracellular content of pS2 protein was constant, suggesting that TPA did not induce secretion of pS2 protein but rather de novo synthesis of the protein. The increase in the pS2 protein content of the medium by TPA was inhibited by simultaneous addition of cycloheximide, but not by that of actinomycin D. Northern‐blot hybridization analysis showed that the amount of pS2 mRNA was unchanged by treatment of the cells with TPA. These results indicate that TPA does not induce transcription of the pS2 gene, and suggest that the main effect of TPA results from the induction of translation of pS2 mRNA.
Transduction of reducing power across the plasma membrane by reduced glutathione
Authors: CIRIOLO Maria R. , PACI Maurizio , SETTE Marco , DE MARTINO Angelo , BOZZI Argante , ROTILIO Giuseppe
Published: March 2005

The NMR signal of reduced glutathione (GSH) was monitored in intact human erythrocytes by the 1H spin‐echo Carr‐Purcell‐Meiboom‐Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte membrane, produced an approximate twofold increase of the GSH signal in glucose‐depleted cells. Addition of oxidised glutathione (GSSG), did not affect the signal, and addition of GSH to hemolysates gave a much smaller increase. Reduction of internal GSSG by NADPH‐dependent enzymes was excluded by experiments with glucose‐supplied or glucose‐6‐phosphate dehydrogenase deficient cells. Involvement of external thiol groups of the erythrocyte membrane was shown by the lack of effect in cells treated with an impermeable thiol‐blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in erythrocytes with variable genetic deficiency of spectrin. Protein‐glutathione mixed disulfides appeared to be the source of the NMR response since an increase of their content, by diamide treatment or aging procedures, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can transduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur‐rich proteins spanning across the erythrocyte membrane.
Multiphasic denaturation of glutathione transferase B1‐1 by guanidinium chloride
Authors: SACCHETTA Paolo , ACETO Antonio , BUCCIARELLI Tonino , DRAGANI Beatrice , SANTARONE Stella , ALLOCATI Nerino , DI ILIO Carmine
Published: March 2005

The unfolding and refolding mechanisms of dimeric glutathione transferase GSTB1‐1 from Proteus mirabilis, using guanidinium chloride as a denaturant, have been investigated. The protein transitions were monitored by enzyme activity, intrinsic fluorescence, far ultraviolet circular dichroism and gel‐filtration chromatography. The non coicidence of denaturation curves at equilibrium indicates that the unfolding of GSTB1‐1 is a multistep process, i. e. inactivation of the structured dimer, dissociation into partially structured monomers followed by complete unfolding. In the 50% inactivated enzyme the Km for glutathione increases threefold, while the kcat appears almost the same, indicating that the initial phase of the denaturation involves the binding site of glutathione. The rapid recovery of the folded dimer precedes the complete enzyme reactivation. This indicates that the reconstitution of the native structure of GSTB1‐1 is the result of folding and association of compact monomers followed by subtle rearrangements of assembled monomers that build up the active site.