bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterisation of d‐glucose oxidase from white‐rot fungus Pleurotus ostreatus
Authors: SHIN Kwang‐Soo , YOUN Hong‐Duk , HAN Young‐Hoon , KANG Sa‐Ouk , HAH Yung Chil
Published: March 2005

d‐Glucose oxidase was purified 27.5‐fold to apparent homogeneity with an overall yield of 23.8%, from Pleurotus ostreatus, through a purification procedure of ammonium sulphate precipitation, gel‐permeation, anion‐exchange and hydrophobic‐interaction chromatography. The molecular mass determined by gel filtration was found to be 290 kDa. SDS/PAGE revealed that the enzyme consists of four subunits with a molecular mass of 70 kDa. The absorption spectra of the enzyme exhibit maxima at 280, 360 and 460 nm. The enzyme shows a fluorescence spectrum with an excitation maximum at 470 nm and an emission maximum at 530 nm. These results indicate that the prosthetic group of the enzyme is flavin and that the enzyme contains 4 mol flavin/mol enzyme. The enzyme is optimally active at 50°C and at pH 5.5–6.0. It exhibits broad affinity for various sugars and specificity for d‐glucose with Km value of 1.34 mM. 2,6‐Dichloroindophenol, Wurster's blue, and 4‐benzoquinone can function as electron acceptors but phenazine methosulphate cannot function as an electron acceptor. The enzyme is inhibited completely by mercuric chloride and partially by silver sulphate, sodium azide and 8‐hydroxyquinoline.
Characterization of the haem environment in Methylophilus methylotrophus ferricytochrome c″ by 1H‐NMR
Authors: COSTA Helena S. , SANTOS Helena , TURNER David L.
Published: March 2005

Two‐dimensional NMR techniques have been used to assign proton resonances in the haem cavity of Methylophilus methylotrophus cytochrome c”, a monohaem protein with bis‐histidinyl ligation which has been shown to couple electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side‐chain protons from four other residues in the haem pocket have also been identified. The data indicate a highly symmetric unpaired‐electron distribution in the haem group, which agrees with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly exposed. To obtain information on the dynamics of the haem environment, measurements of the 1H/2H‐exchange rates of amide protons located in the haem cavity were performed. The two faces of the haem are found to differ markedly with respect to water accessibility. All of this information, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and that the redox‐linked protonation occurs via a channel running through the haem cleft on the opposite face.
Effect of cell attachment and growth on the synthesis and fate of dolichol‐linked oligosaccharides in Chinese hamster ovary cells
Authors: CACAN René , LABIAU Odette , MIR Anne‐Marie , VERBERT André
Published: March 2005

The inhibition of cellular processes in suspended anchorage‐dependent Chinese hamster ovary (CHO) cell lines and their restoration upon attachment to a solid substrate has been used as a model to study the relationship between oligosaccharide‐diphospho‐dolichols and their metabolic products (glycoprotein and soluble oligosaccharide material, i. e. oligosaccharide phosphates and neutral oligosaccharides). Using metabolic labelling we demonstrated that suspended cells have a low incorporation rate into lipid intermediates and into glycoproteins. The oligosaccharide‐lipid populations are mainly glucosylated and the neutral oligosaccharides have exclusively a chitobiosyl residue at their reducing end. In contrast, monolayer cells exhibit a high incorporation rate into lipid intermediates with a pattern dominated by two species containing either two or five mannose residues, and into glycoproteins with a pattern similar to the one observed for suspended cells (i.e. glucosylated species). In monolayer cells the neutral oligosaccharides possess either one or two GlcNAc residues at their reducing end. The variations in the nature and in the quantity of soluble oligosaccharide material as a function of the cell density reflects regulatory points in the synthesis of N‐glycosyl proteins. The first regulatory point could be the control of the quantity of non‐glucosylated oligosaccharide‐lipids to be channelled toward the glucosylated lipid‐donor pool. The level of this donor pool being constant, the oligosaccharidetransferase could utilize oligosaccharide‐lipid donors at a constant rate by two different reactions: either transfer onto protein when acceptors are available, or transfer onto water generating neutral oligosaccharides possessing two GlcNAc residues at the reducing end. Another regulatory point would be the degradation of a part of neoglycoproteins leading to the release of neutral oligosaccharides possessing one GlcNAc residue at the reducing end.
An arrestin homolog of blowfly photoreceptors stimulates visual‐pigment phosphorylation by activating a membrane‐associated protein kinase
Authors: BENTROP Joachim , PLANGGER Anette , PAULSEN Reinhard
Published: March 2005

An arrestin homolog (Arr2, 49‐kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light‐dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well‐defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin‐agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45–49 kDa. The isoelectric point, as judged by gel isolelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites.

Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light‐induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin‐stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual‐pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.
Regulation of rabbit α1‐acid glycoprotein gene expression in acute‐phase liver
Authors: RAY Bimal K. , GAO Xiong , RAY Alpana
Published: March 2005

To identify the cis‐acting DNA sequences responsible for inducible transcription of rabbit α1‐acid glycoprotein gene, 5′‐flanking region containing 529 bp of this gene and its various 5′‐deletions were linked to the reporter gene coding for the bacterial chloramphenicol acetyltransferase (CAT) and analyzed for their ability to confer cytokine‐mediated inducibility to the reporter CAT gene in liver cells. Deletion analysis has identified a 151‐bp region from the sequence – 186 to –35, that contains the regulatory promoter element(s) responsible for stimulation mediated by cytokines present in the conditioned‐medium. Using mobility shift assays, we have identified highly inducible nuclear factors in acute liver nuclear extract that interact with this regulatory promoter region. DNase I footprint analysis has revealed two adjacent nuclear factor binding sites and competition of DNA‐binding activity has indicated that the distal element of these two sites has higher affinity for nuclear factors than the proximal one. Both of these two regions have been found to be capable of directing conditioned‐medium‐induced transcription. Studies on the characterization of nuclear factors binding to these elements have shown that they belong to a class of transcription factors called CCAAT‐enhancer binding protein (C/EBP). Our results indicate that binding of C/EBP‐like factors to the inducible promoter elements of rabbit α1‐acid glycoprotein gene is highly specific and the induction of this gene under acute‐phase conditions may involve their participation.
Secondary structure of the DNA‐binding domain of the c‐Myb oncoprotein in solution
Authors: JAMIN Nadége , GABRIELSEN Odd S. , GILLES Nicolas , LIRSAC Pierre‐Noël , TOMA Flavio
Published: March 2005

The DNA‐binding domain of the c‐Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix‐turn‐helix(HTH) motif. As a first step towards determination of the three‐dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly 15N‐ labeled and 13C, 15N double‐labeled R2R3 and, a selectively 15N‐enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 1H, 15N and 13Ca atoms and determine the secondary structure of R2R3 in solution.

The R3 repeat is composed of three helices (residues 62–75, 78–85 and 91–100) while for the R2 repeat only two helices are found (residues 10–23 and 28–34). The remaining C‐terminal part of the R2 repeat, predicted to be helical and part of the HTH motif, undergoes intermediate conformational exchange processes. Stabilization of this segment might occur upon binding to DNA.
Secretory phospholipase A2 is not required for arachidonic acid liberation during platelet activation
Authors: MOUNIER Carine , FAILI Ahmad , VARGAFTIG B. Boris , BON Cassian , HATMI Mohamed
Published: March 2005

The subcellular localization of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in resting and activated platelets, and their involvement in arachidonic acid liberation during platelet activation, were studied. The amounts of sPLA2 and cPLA2 recovered were not modified during platelet activation. sPLA2 was mainly associated with the organelles of resting platelets (71% of total activity) and was released into the extracellular medium during cell activation (60% of total activity), whereas the majority of cPLA2 was localized in the cytosol of resting and activated platelets. The secretion of sPLA2 correlated with the release of ATP. sPLA2‐depleted platelets aggregated as much as control platelets and produced similar amounts of thromboxane B2 upon thrombin activation. These results indicate that sPLA2 is not involved in the liberation of arachidonic acid during platelet activation.
Restriction‐enzyme cleavage of DNA modified by platinum(II) complexes
Authors: BRABEC Viktor , BALCAROVA Zdenka
Published: March 2005

The effect of binding of cis‐diamminedichloroplatinum(II), its trans isomer and diethylenetriami‐nechloroplatinum(II) chloride to DNA on the splicing effectiveness of BamHI, EcoRI and SalI restriction endonucleases has been determined by means of gel electrophoresis. All three platinum complexes inhibit the cleavage of linearized plasmid DNA. In addition, the three platinum complexes bound to DNA constitute a barrier across which the linear diffusion of EcoRI on DNA is difficult. We interprete these findings to mean that the splicing effectiveness of restriction enzymes is influenced by bifunctional and monofunctional DNA adducts of platinum via both steric interference and DNA conformational distortions. Whereas the platinum adducts in the restriction sites or in their very close proximity inhibit the cleavage, the lesions occurring a greater distance from the restriction site can slow down the process of the localization of recognition sequences.
High expression in Escherichia coli of the gene coding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii
Authors: BLECHER Oshra , GOLDMAN Sarah , MEVARECH Moshe
Published: March 2005

The gene coding for the enzyme dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii was recombined into the Escherichia coli expression vector pET11d. Following induction, the enzyme was produced in large quantities and accumulated in the cells in an insoluble form. The enzymic activity could be efficiently reconstituted by dissolving the aggregate in 6 M guanidine hydrochloride followed by dilution into salt solutions. Mutants were produced in which Lys30 was converted to Leu (K30L), Lys31 was converted to Ala (K31 A) and a double mutant in which both lysines were converted (K30L, K31 A). The mutated enzymes were produced in E. coli, activated and purified to homogeneity. The effect of the salt concentration on the steady‐state kinetic parameters was determined. It was found that the salt concentration affects the Km but not kcat of the various mutants.
A six‐armed, tenascin‐like protein extracted from the Porifera Oscarella tuberculata (Homosclerophorida)
Authors: HUMBERT‐DAVID Nathalie , GARRONE Robert
Published: March 2005

A six‐armed complex could be extracted from the marine sponge Oscarella tuberculata by a two‐step incubation, first in Tris‐buffered saline containing EDTA, then in Tris‐buffered saline containing urea. The crude extracts contained, in addition, collagen fibrils with surface filaments, individual filaments resembling collagen molecules, and laminin/nidogen‐like complexes. The extracts were subsequently purified by gel‐filtration chromatography and low‐pressure ion‐exchange chromatography on DEAE‐cellulose, then analyzed by SDS/PAGE and immunoblotting methods. A glycoprotein of high molecular mass was isolated, and reduced to subunits of 230 kDa. After transfer to nitrocellulose, both the complex and its subunits were faintly stained by antibodies against amphibian tenascin. Electron microscopy of the purified extracts demonstrated the presence of a large population of tenascin‐like molecules and complexes of several molecules interacting with each other by their central globule.
Both isoforms of protein phosphatase Z are essential for the maintenance of cell size and integrity in Saccharomyces cerevisiae in response to osmotic stress
Authors: HUGHES Valerie , MÜLLER Anneliese , STARK Michael J. R. , COHEN Patricia T. W.
Published: March 2005

The sequences of two genes encoding the protein‐serine/threonine‐phosphatases PPZ1 and PPZ2 from Saccharomyces cerevisiae have been determined. The molecular masses of PPZ1 and PPZ2 are 77.5 and 78.5 kDa, respectively, and each protein consists of two distinct domains. The C‐terminal half of each molecule is 93% identical in PPZ1 and PPZ2, and comprises the protein‐phosphatase catalytic domain, while the N‐terminal halves, which are rich in serine and asparagine (PPZ1) or serine and arginine (PPZ2), are only 43% identical. Both N‐termini start with the amino acids Met‐Gly‐Asn, suggesting that after removal of the initiating methionine, the N‐terminal glycine of the mature protein is myristoylated. Disruption of the gene encoding either PPZ1 or PPZ2 leads to an increase in cell size and cell lysis, the latter being more pronounced in cells disrupted in PPZ1. Haploid cells carrying a double disruption of PPZ1 and PPZ2 genes also show a marked increase in cell size and cell lysis, which can be significantly reduced by the addition of 1 M sorbitol to the growth medium. These results suggest that PPZ1 and PPZ2 play a role in regulating osmotic stability.
Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis
Authors: KUIPERS Oscar P. , BEERTHUYZEN Marke M. , SIEZEN Roland J. , DE VOS Willem M.
Published: March 2005

The nisin gene cluster nisABTCIPR of Lactococcus lactis, located on a 10‐kbp DNA fragment of the nisin‐sucrose transposon Tn5276, was characterized. This fragment was previously shown to direct nisin‐A biosynthesis and to contain the nisP and nisR genes, encoding a nisin leader peptidase and a positive regulator, respectively [van der Meer, J. R., Polman, J., Beerthuyzen, M. M., Siezen, R. J., Kuipers, O. P. & de Vos, W. M. (1993) J. Bacteriol. 175, 2578–2588]. Further sequence analysis revealed the presence of four open‐reading frames, nisB, nisT, nisC and nisI, downstream of the structural gene nisA. The nisT, nisC and nisI genes were subcloned and expressed individually in Escherichia coli, using the T7‐RNA‐polymerase system. This resulted in the production of radio‐labelled proteins with sizes of 45 kDa (NisC) and 32 kDa (NisI). The nisT gene product was not detected, possibly because of protein instability. The deduced amino acid sequence of NisI contained a consensus Iipoprotein signal sequence, suggesting that this protein is a lipid‐modified extracellular membrane‐anchored protein. Expression of nisI in L. Iactis provided the cells with a significant level of protection against exogeneously added nisin, indicating that NisI plays a role in the immunity mechanism. In EDTA‐treated E. coli cells, expression of nisI conferred up to a 170‐fold increase in immunity against nisin A compared to controls. Moreover, a lactococcal strain deficient in nisin‐A production, designated NZ9800, was created by gene replacement of nisA by a truncated nisA gene and was 10‐fold less resistant to nisin A than the wild‐type strain. A wild‐type immunity level to nisin and production of nisin was obtained in strain NZ9800 harboring complementing nisA and nisZ plasmids. Transcription analyses of several L. IIactis strains indicated that an expression product of the nisA gene, together with NisR, is required for the activation of nisA transcription.
Expression of the laminin‐A chain is down‐regulated by a non‐canonical polyadenylation signal
Authors: GOTTSCHLING Claudia , HUBER Johann , OBERBÄUMER Ilse
Published: March 2005

It is well accepted that 3′ untranslated regions (UTR) are an essential part of mRNA. However, little is known in detail about the contribution of different regions of 3′UTR on synthesis, stability and translatability of their mRNA. In addition to the highly conserved hexanucleotide AAUAAA, some consensus sequences for 3′‐end processing and polyadenylation have been characterized, but most of this work has been done with viral mRNA or β‐globin mRNA. We have studied the influence of the 3′UTR of the mRNA for the three chains A, B1, B2 of laminin on the expression of a reporter gene(galK). I aminin is a large glycoprotein of basement membrances and all three polypeptide chains are needed in equal amounts for a functional molecule. The three 3′UTR of the laminin mRNA differ widely with respect to length, number of polyadenylation signals and other consensus sequences. Nevertheless, all three 3′UTR reduce the expression of the reporter gene at least three‐fold, when the corresponding cDNA sequences are inserted downstream of the reporter gene instead of the 3′UTR of simian virus 40 early genes. The 3′UTR of laminin‐A mRNA contains the non‐canonical polyadenylation signal AUUAAA which seems to be responsible for the limiting amounts of laminin‐A mRNA and protein compared to those for laminin B1 and B2 [Speth and Oberbäumer (1993) Exp. Cell Res. 204, 302–310]. Mutation of the laminin‐A polyadenylation signal to the canonical form AAUAAA increases expression by a factor of 2.5.
Molecular cloning and expression of Galβ1,3GalNAcα2,3‐sialyltransferase from mouse brain
Authors: LEE Young‐Choon , KUROSAWA Nobuyuki , HAMAMOTO Toshiro , NAKAOKA Takashi , TSUJI Shuichi
Published: March 2005

DNA clones encoding β‐galactoside α2,3‐sialyltransferase have been isolated from mouse brain cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence revealed an open reading frame coding for 337 amino acids, and the deduced amono acid sequence showed 80% identity with that of porcine submaxillary gland Galβ1,3GalNAc α2,3‐sialyltransferase. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2‐terminal cytoplasmic domain, a signal‐membrane anchor domain, a proteolytically sensitive stem region, and a large COOH‐terminal active domain. The identity of this enzyme was confirmed by construction of a recombinant sialyltransferase in which the NH2‐terminal part including the cytoplasmic tail, signal‐anchor domain and stem region was replaced with an immuno‐globulin signal sequence. The expression of this recombinant in COS‐7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. This enzyme exhibited the transferase activity toward only the disaccharide moiety of Galβ1,3GalNAc of glycoproteins and glycolipids, no significant activity being detected for the other substrates tested.
A novel 77‐residue peptide from porcine brain contains a leucine‐zipper motif and is recognized by an antiserum to delta‐sleep‐inducing peptide
Authors: SILLARD Rannar , SCHULZ‐KNAPPE Peter , VOGEL Petra , RAIDA Manfred , BENSCH Klaus W. , FORSSMANN Wolf‐Georg , MUTT Viktor
Published: March 2005

In 1977 a nonapeptide, called delta‐sleep‐inducing peptide (DSIP) was characterized in rabbit cerebral venous blood plasma during thalamic stimulation to induce sleep. Evidence for the existence of DSIP in the central nervous system and in numerous peripheral organs of various mammalian species has been obtained using immunochemical techniques. Later findings have revealed the existence of large forms of DSIP‐like immunoreactivity. We decided to investigate the molecular identity of such large forms of DSIP‐like immunoreactivity by direct isolation. We have purified and characterized using amino acid analysis, sequencing, mass spectrometry and radioimmunoassay a 77‐residue peptide, denoted DIP (DSIP‐immunoreactive peptide), from an acid extract of porcine brain. DIP is recognized by an antiserum raised against synthetic rabbit DSIP. The amino acid sequence of DIP, however, is not related to that of DSIP, but it contains a putative leucine‐zipper motif, a proline/glutamic‐acid‐rich domain, three potential phosphorylation sites and exhibits an acetylated N‐terminus. The N‐terminal but not the C‐terminal part of the newly isolated peptide shares clear homology with the sequence of a protein induced by transforming growth factor β1 and other growth factors in mouse osteoblastic cells. DIP is also structurally similar to a baculoviral protein p10. The function of DIP remains unclear but its involvement in transcriptional regulation is probable.
The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase
Authors: VOLPE Filippo , BALLANTINE Stuart P. , DELVES Chris J.
Published: March 2005

The nucleotide sequence of a folic acid synthesis (fas) gene from Pneumocystis carinii contains an open reading frame (ORF) that predicts a protein of 740 amino acids with an Mr of 83979. A recombinant baculovirus was constructed which directed expression of the predicted Fas 740 polypeptide in cultured Spodoptera frugiperda (SF9) insect cells. The overexpressed ‘full‐length’ protein migrated anomalously in sodium dodecyl sulfate/polyacrylamide gels, with an apparent molecular mass of 71.5 kDa. An abundant 69‐kDa species was also recognized by polyclonal sera specific for the Fas protein in immunoblotting analyses. Dihydroneopterin aldolase, dihydropterin pyrophosphokinase and dihydropteroate synthase activities were readily detected in SF9 extracts in which the 71.5/69‐kDa immunoreactive species were overproduced, demonstrating that three enzyme functions involved in catalysing three sequential steps of the folate biosynthetic pathway are encoded by a single gene in P. carinii. Importantly, the polyclonal sera recognize a single 69‐kDa species in P. carinii extracts suggesting that the three activities are indeed properties of a single polypeptide, although the nature of the suggested post‐translational modification is unknown. Location of the individual enzyme domains with the Fas polypeptide based upon amino acid sequence similarity to their bacterial counterparts is discussed. Futhermore, expression of various truncated fas gene constructs demonstrates that the complete fas ORF, including the N‐terminus of the predicted polypeptide (FasA domain) whose enzyme function is unknown, must be expressed for maximum dihydroneopterin aldolase (FasB domain) and dihydropteroate synthase (FasD domain) activites. This suggests interactions between the domains within the larger polypeptide to stabilize the functions of these two enzymes. The FasC domain, which contains 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase activity, is able to fold and function independently of the other domains. The requirement by mammalian cells for preformed folates, and the absence of dihydroneopterin aldolase, 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase and dihydropteroate synthase from these tissues opens up the possibility of designing higly selective drugs which inhibit these unique targets.
A glutathione S‐transferase with glutathione‐peroxidase activity from Arabidopsis thaliana
Authors: BARTLING Dieter , RADZIO Renate , STEINER Ulrike , WEILER Elmar W.
Published: March 2005

A full‐length cDNA clone for a novel glutathione S‐transferase was isolated from Arabidopsis thaliana and characterized. The cDNA encodes a polypeptide of 218 amino acids with a calculated molecular mass of 24363 Da. The sequence was most related to the theta class within the glutathione‐S‐transferase superfamily of enzymes. The protein encoded by the cDNA was functionally expressed and enzymically active in Escherichia coli; glutathione‐S‐transferase activity with the standard enzyme substrate 1‐chloro‐2,4‐dinitrobenzene was demonstrated (apparent Km, 10mM; apparent Km for glutathione, 0.08 mM). The enzyme is substrate specific and did not use several electrophilic reduced‐glutathione acceptor molecules for conjugation. However, it efficiently catalyzed the conversion of 13‐hydroperoxy‐9,11,15‐octadecatrienoic acid (Km, 0.67mM) as well as 13‐hydroperoxy‐9,11‐octadecadienoic acid (Km, 0.79mM) to the corresponding hydroxy derivatives with concomitant formation of oxidized glutathione. The enzyme did not use H2O2 as substrate. Thus, the cloned A. thaliana enzyme functions as glutathione peroxidase and, in the plant cell, may be involved in the removal of reactive organic hydroperoxides, such as the products of lipid peroxidation. The enzyme is structurally and enzymically, however, unrelated to the selenium‐containing glutathione peroxidases. Enzymic and immunoblotting data suggest that the A. thaliana enzyme is soluble and constitutively expressed in vegetative rosettes, but is under developmental control during the transition to bolting and flowering.
Peptidylproline cis‐trans‐isomerases: immunophilins
Authors: GALAT Andrzej
Published: March 2005

Two sequence‐unrelated families of proteins possess peptidylproline cis‐trans‐isomerase activities (PPlase). PPlases are highly sequence conserved and multifunctional proteins which are present in many types of cells with a considerably divergent phylogenetic distribution. On the cellular level, PPlases occur in every compartment, both as free species and anchored to membranes. Diverse posttranslational modifications such as glycosylation, N‐terminal modifications and phosphorylation constitute the additional functional features of PPlase. Folding, assembly and trafficking of proteins in the cellular milieu are regulated by PPlase. These enzymes accelerate the rate of in‐vitro protein folding and they have the ability to bind proteins and act as chaperones. Some PPlases are coregulatory subunits of molecular complexes including heat‐shock proteins, glucocortcoid receptors and ion channels. Secreted forms of PPlases are inflammatory and chemotactic agents for monocytes, eosinophils and basophils. The potent and clinically useful immunosuppressants CsA, FK506 or rapamycin bind with high affinities to PPlases (immunophilins). The binding criterion allows us to sort the PPlases for the following two superfamilies of proteins: the cyclophilins (CsA‐binding proteins) and the FKBP (FK506/rapamycin‐binding proteins).

Although none of PPlases appeared to be essential for the viability of haploid yeast cells some of the immunophilin/immunosuppressant complexes are toxic both for yeast and mammalian cells. At least seven unlinked genes of cyclophilins and four unlinked genes of FKBP exist in human genomic DNA. Selected immunophilins regulate two different signalling pathways in lymphoid cells, namely the secretion of growth factors by stimulated T‐cells and interleukin‐2‐induced T‐cell proliferation. Moreover, selected FKBP mediate the cytotoxic effects of rapamycin in non‐lymphoid cells. Accounts of the discovery of PPlases (immunophilins) and their functions are given in this review. A larger spectrum of proteins is analysed in relation to various signal‐transduction pathways in lymphoid cells which involve immunophilins or their complexes with the immunosuppressants CsA, FK506 or rapamycin.
Selective inhibition of N‐acetylglucosamine and galactose‐specific lectins including the 14‐kDa vertebrate lectin by novel synthetic biantennary oligosaccharides
Authors: GUPTA Dipti , SABESAN Subramaniam , BREWER C. Fred
Published: March 2005

A novel series of synthetic bantennary tri‐, penta‐ and hepta‐saccharides with terminal β‐GlcNAc, β‐LacNAc and αNeuAc(2,6)βLacNAc residues, respectively, [LacNAc = Galβ(1,4)GlcNAc] connected to a core Gal residue were evaluated for their inhibitory potencies for specific plant and animal lectins. Six isomeric trisaccharides with two β‐GlcNAc residues at the 2,3‐, 2,4‐, 2,6‐, 3,4‐, 3,6‐, or 4,6‐positions of the core Gal were tested for their hemagglutination inhibition activities aganist two GlcNAc‐specific lectins, Griffonia simplicifolia II (GS II) and wheat germ agglutinin (WGA), The 2,3‐, 2,4‐, 2,6‐ and 3,6‐trisaccharides inhibited WGA 12–50 times more strongly than GlcNAc, whereas only weak or no inhibition was observed with GS II. The 3,4‐ and 4,6‐trisaccharides did not inhibit either of the lectins. Six biantennary isomeric pentasaccharides containing two terminal β‐LacNAc residues with branching patterns similar to the trisaccharides showed selective hemagglutination inhibition of five Gal/GalNAc‐specific plant lectins and the 14‐kDa Gal‐specific calf spleen lectin. The plant lectins include the soybean agglutinin (SBA), ricin agglutinin‐I (RCA‐I), and three Erythrina lectins with similar specificities; Erythrina indica (EIL), E. corallodendron (ECorL), and E. cristagalli (ECL). The 2,3‐pentasaccharide inhibited only SBA and the 14‐kDa lectin, and thus was a selective inhibitor among the plant lectins. The 2,6‐pentasaccharide inhibited SBA, ECL and the 14‐kDa lectin, but not RCA‐l or the two other Erythrina lectins. The 4,6‐pentasaccharide did not inhibit any of the plant lectins, but was a specific inhibitor of the 14‐kDa calf spleen lectin. Synthetic heptasaccharides analogs with 2,4‐, 2,6‐, 3,6‐ and 4,6‐branching patterns and terminal α(2,6)NeuAc residues all showed 25‐fold stronger inhibition against the α(2,6)sialicacid‐specific elderberry (Sambucus nigra L.) bark lectin as compared to a monovalent disaccharide αNeuAc(2,6)βGalOR. The lack of inhibition. of αNeuAc(2,6)βGalOR derivatives methylated at the C6 of the Gal residue and a sulfur‐linked thiosialoside derivative demonstrates that the 2,6‐anomeric linkage region is important for lectin recognition. Selective inhibition of the Gal/GalNAc‐specific lectins was observed for two isomeric C6 methyl‐substituted Gal derivatives of methyl β‐LacNAc which possess different preferred rotamer orientations about the C5‐C6 bond of the Gal residue.
Purification of trehalose synthase from baker's yeast
Authors: LONDESBOROUGH John , VUORIO Outi E.
Published: March 2005

A trehalose synthase purified from baker's yeast contained 56‐kDa, 102‐kDa and 123‐kDa polypeptides as its main components. The 102‐kDa polypeptide was isolated and shown to be a specific trehalose‐6‐phosphatase. The trehalose‐6‐phosphate synthase (Tre6P synthase) activator described by Londesborough and Vuorio [(1991) J. Gen. Microbiol. 137, 323–330] was shown to be phosphoglucoisomerase and to function entirely by generating fructose 6‐phosphate. Below 35°C, fructose 6‐phosphate is a powerful activator of the Tre6P synthase activity of intact trehalose synthase, especially at physiological phosphate concentration, but does not affect its trehalose‐6‐phosphatase activity nor the Tre6P synthase activity of truncated trehalose synthase containing truncated versions of the 123‐kDa polypeptide. At 50°C, activation by fructose 6‐phosphate and inhibition by phosphate are greatly decreased, resulting in an unusually high temperature‐dependence for the Tre6P synthase activity at a physiological phosphate concentration (2 mM).
Structural study of a cell‐wall mannan of Saccharomyces kluyveri IFO 1685 strain
Authors: SHIBATA Nobuyuki , KOJIMA Chizuko , SATOH Yohko , SATOH Richi , SUZUKI Atsuko , KOBAYASHI Hidemitsu , SUZUKI Shigeo
Published: March 2005

Acetolysis of the cell‐wall mannan of Saccharomyces kluyveri under mild conditions, gave fragments with 1–6 mannose residues. The structures of mannopentaose and mannohexaose were determined to be

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and

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respectively, by two‐dimensional homonuclear Hartmann‐Hahn spectroscopy and a sequential NMR assignment method that combines 1H‐13C correlated spectroscopy, relayed coherence transfer spectroscopy, 1H‐detected heteronuclear multiple‐bond connectivity and methylation analysis. The H1 proton chemical shift of a neighboring α‐1,2‐linked mannose unit of the 3‐O‐substituted structure was shifted upfield by the addition of a mannose unit to the adjacent 3 O‐substituted unit by an α‐1,6 linkage. The characteristic H1–H2‐correlated cross‐peak of the α‐1,3‐linked mannose unit substituted by a β‐1,2 linkage, β1→2Manα1→3, in the mannan of S. kluyveri, as also found by two‐dimensional homonuclear Hartmann‐Hahn spectroscopy in the mannan of Candida guilliermondii, a pathogenic yeast in man.
Cloning from a mouse osteoblastic cell line of a set of transforming‐growth‐factor‐β1‐regulated genes, one of which seems to encode a follistatin‐related polypeptide
Authors: SHIBANUMA Motoko , MASHIMO Jun'ichi , MITA Akira , KUROKI Toshio , NOSE Kiyoshi
Published: March 2005

Transforming growth factor(TGF)β1 is a potent inhibitor of growth in mouse osteoblastic MC3T3‐E1 cells. To isolate genes that are induced by TGFβ1, the differential screening method was adopted using a cDNA library constructed from cells treated with TGFβ1 for 4 h. Six independent cDNA clones were isolated (TGFβ‐stimulated clone, TSC‐5, TSC‐36, TSC‐115, TSC‐128, TSC‐160 and TSC‐161), the expression of which was increased by TGFβ1‐treatment with maximal expression at 6–10 h. The steady‐state levels of TSC‐36, TSC‐128 and TSC‐160 increased almost tenfold, whereas those of TSC‐5, TSC‐115 and TSC‐161 were elevated at most threefold. From partial nucleotide sequences, TSC‐160 was found to be identical to rrg (ras‐recision gene, lysyl oxydase), and TSC‐115 had 80% similarity with tropomyosin cDNA, whereas other genes seemed novel. Expression of TSC‐36 and TSC‐160 was dramatically decreased in v‐Ki‐ras‐transformed MC3T3 cells or in transformed NIH 3T3 cells (DT), and was recovered to normal levels in a flat revertant (C11). A nearly full‐length copy of TSC‐36 cDNA was isolated, and an open reading frame indicated that it encodes a protein of 35 kDa. An antiserum was raised against the C‐terminal peptide predicted from the nucleotide sequence, and a polypeptide with an approximate molecular mass of 38 kDa was detected in cultured medium of MC3T3‐E1 cells. The amino acid sequence of TSC‐36 protein was found to have some similarity with follistatin, an activin‐binding protein, and a limited similarity with the secreted protein rich in cysteine (SPARC).
Gly85 to Val substitution in proα1(I) chain causes mild osteogenesis imperfecta and introduces a susceptibility to protease digestion
Authors: VALLI Maurizia , ZOLEZZI Francesca , MOTTES Monica , ANTONIAZZI Franco , STANZIAL Franco , TENNI Ruggero , PIGNATTI PierFranco , CETTA Giuseppe
Published: March 2005

In this paper we describe a mild moderate form of osteogenesis imperfecta caused by a point mutation in COL1A1 which converted glycine 85 to valine.

The valine substitution introduced into the triple‐helical domain of type‐I collagen a conformational perturbation causing susceptibility to digestive proteases. In fact, SDS/PAGE of pepsin‐treated collagen showed the presence of a faint band, migrating between α1(I) and α2(I), both in the medium and in the cell layer. On trypsin digestion the band, a shortened form of α1(I), had a melting temperature of 39.5°C. If the triple‐helical collagen was obtained after trypsin or chymotrypsin digestion of procollagen, two shortened bands were identified; the enzymes cleaved about 40% of the trimers. The mutant procollagen was normally secreted and processed in the extracellular matrix at a normal rate. When native type‐I collagen was formed after dextran‐sulfate incubation, only chains of normal length were found, suggesting that the fibroblast proteases did not recognize the alteration introduced by the mutation.

The effects of glycine 85 to valine substitution are compared with those produced by a previously described arginine substitution of the same residue (Deak et al., 1991).
Sorbitol dehydrogenase
Authors: WEN Yi , BEKHOR Isaac
Published: March 2005

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis λZAP II cDNA library. The full‐length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42‐amino‐acid polypeptide linked to the N‐terminal amino acid of the enzyme, generating a pre‐sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre‐SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post‐translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3′ untranslated region of the cDNA contained a non‐coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362–1367, the second at nucleotides 1465–1470, and the third at nucleotides 2212–2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957–960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern‐blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3–2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full‐length cDNA.
In vitro binding of nuclear proteins to the barley plastocyanin gene promoter region
Authors: NIELSEN Peter S. , GAUSING Kirsten
Published: March 2005

Plastocyanin is a nuclear‐encoded chloroplast protein participating in electron transport during photosynthesis. The plastocyanin gene is expressed in photosynthetic tissue in a developmentally regulated manner and the expression is stimulated by light. A genomic clone encoding the plastocyanin precursor was isolated from a barley (Hordeum vulgare) λ library using a barley cDNA clone as a probe and the sequence of a 1.9‐kb DNA fragment containing the plastocyanin gene was determined. TATA and CCAAT boxes are located 34‐bp and 68‐bp, respectively, upstream of the transcription start site, the 5′‐untranslated leader is 78 nucleotides long, and the intronless gene has at least two different polyadenylation sites.

DNA sites in the plastocyanin gene that mediate binding of barley nuclear proteins were mapped by mobility‐shift assays with fragments of the promoter/upstream region. Two of the three specific binding sites characterised in more detail were found to form complexes with the same factor in cross‐competition experiments. One of these sites, narrowed down to a 17‐bp sequence at position –512, contains the consensus binding site for Myb‐like transcription factors. The third specific binding site, located at position –622, contains the sequence CACGTG which is a high‐affinity‐binding site for transcription factors of the basic‐region leucine‐zipper family.
Cloning, DNA sequence analysis and partial characterization of pepN, a lysyl aminopeptidase from Lactobacillus delbrückii ssp. lactis DSM7290
Authors: KLEIN Jürgen Robert , KLEIN Ulrike , SCHAD Michael , PLAPP Roland
Published: March 2005

In cell extracts of Lactobacillus delbrückii ssp. lactis DSM7290 a peptidase with the ability to hydrolyse Phe‐β‐naphthylamide (Phe‐β‐NA) and His‐β‐NA could be detected. Escherichia coli lacking the enzyme activity in an enzymic plate assay was used to screen high‐copy‐number and low‐copy‐number plasmid libraries of size‐fractionated Lactobacillus DNA. Clones with the desired phenotype were detected, and the gene, designated pepN, was further subcloned and sequenced. A large open reading frame of 2529 nucleotides is predicted to encode a protein of 843 amino acids (95358 Da). Comparison of the pepN gene from Lb. delbrückii ssp. lactis DSM7290 indicates that it is homologous to genes of the family of Zn2+‐metallohydrolases and PepN shows identity with the active centre Zn2+‐binding motif of these enzymes. The substrate Lys‐β‐NA is more effectively cleaved than Phe‐β‐NA or His‐β‐NA which were used for screening in E. coli. The cloned pepN gene was efficiently overexpressed in E. coli and subcloning of the gene in Lactobacillus casei resulted in a moderate overexpression of approximately 20‐fold. The pepN gene product was purified from the pepN‐deficient E. coli strain CM89, using the substrate Lys‐p‐nitroanilide (Lys‐NH‐Ph) in the assay procedure. In a four‐step procedure including streptomycin sulfate precipitation, anion‐exchange chromatography and gel filtration the peptidase was purified to electrophoretic homogeneity.
Further characterization of structural and electric properties of non‐spherical α‐crystallin
Authors: VAN HAERINGEN Bart , van Den BOGAERDE Marco R. , EDEN Don , VAN GRONDELLE Rienk , BLOEMENDAL Michael
Published: March 2005

Recently we have shown that the population of native α‐crystallin, isolated using size‐exclusion chromatography from eye lenses of calves, is multimodal. Most of the protein probably possesses an almost spherical appearance, but at least one of the other modes represents more extended, ellipsoidally and/or cylindrically shaped molecules [Van Haeringen, B., Eden, D., Van den Bogaerde, M. R., Van Grondelle, R. & Bloemendal, M. (1992) Eur. J. Biochem. 210, 211–216]. In the present study, we characterize various subpools of a single α‐crystallin size‐exclusion chromatography elution peak by means of transient‐electric‐birefringence measurements, ultraviolet linear‐dichroism spectroscopy and analytical fast protein liquid chromatography. It is concluded that the fractions have a well‐defined stable mass and are not in reversible equilibrium with each other. All pools appear to be composed of at least two types of differently shaped molecules. The hydrodynamic dimensions and electric properties of the different α‐crystallin species are characterized. The non‐spherical α‐crystallin is found to be optically and electrically more anisotropic, and to contain a larger permanent electric dipole moment than the spherical form. A model for the composition of the α‐crystallin pool is presented.
Cloning, site‐specific mutagenesis, expression and characterization of full‐length chloroplast NADP‐malate dehydrogenase from Pisum sativum
Authors: RENG Winfried , RIESSLAND Robin , SCHEIBE Renate , JAENICKE Rainer
Published: March 2005

Chloroplast NADP‐dependent malate dehydrogenase is regulated by a dithiol redox reaction. The assignment of the groups involved, requires the primary structure of the enzyme to be known. Using the polymerase chain reaction and the cDNA library of Pisum sativum, the sequence of the enzyme and its targeting signal was determined. The gene was cioned in Escherichia coli JM83 and expressed in E. coli JM83 and E. coli B at high yield. The determination of the physical properties of the gene product proves the recombinant protein to be indistinguishable from the enzyme purified from the plant. This holds true, in spite of the fact that the plant enzyme lacks 11 N‐terminal residues. The lengths of the complete polypeptide chain of the recombinant enzyme and its transit peptide are 388 and 53 residues, respectively.

The comparison of the sequences of the mature enzyme with those of known chloroplast NADP‐MDH shows 83–95% identity, but with mitochondrial or bacterial MDH only approximately 20%.

Reduction of the (inactive) oxidized enzyme with dithiothreitol allows mimicking of the in vivo activation. The reaction follows a consecutive second‐order‐kinetics mechanism. Guanidinium chloride (GdmCl) at concentrations below 0.4 M leads to a significant activation of the oxidized form of the enzyme. At [GdmCl] = 0.4–0.46 M, both oxidized and reduced NADP‐MDH show highly cooperative changes in the hydrodynamic and spectral properties, indicating the synchronous breakdown of the quaternary, tertiary and secondary structures.

Site‐directed mutations C23A and C28A do not quench the regulatory properties of the enzyme; additional substitution of alanine for Cys206 and Cys376 renders the enzyme equally active in both the reduced and the oxidized state. Therefore, one can consider these residues, either alone or in combination with Cys23 and Cys28, as responsible for enzyme activation.
Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+‐ATPase
Authors: GIRARDET Jean‐Luc , BALLY Isabelle , ARLAUD Gérard , DUPONT Yves
Published: March 2005

The amino acid sequences of several P‐type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the ‘hinge’ domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca2+‐ATPase (SERCA‐1), this region is located at residues 700–712.

Comparison of the sequence in this domain with calcium‐binding proteins reveals similarities with the center of the helix‐loop‐helix EF‐hand structure that is known to form divalent‐cation‐binding sites. A 38‐residue polypeptide, corresponding to the domain 682–719 of the Ca2+‐ATPase was synthesized and tested for its ability to bind divalent cations.

Circular‐dichroism, intrinsic‐fluorescence and fluorescence‐energy‐transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg2+, with an affinity of 10–15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the α‐helix promoter 2,2,2‐trifluoroethanol.

Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties.

It is proposed that this sequence is involved in the co‐ordination of a Mg2+ in the nucleotide‐binding site and/or in the phosphorylation site of P‐type ATPases.