bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the prophenoloxidase activating enzyme of the freshwater crayfish, Pacifastacus leniusculus
Authors: Wang Ruigong , Lee So Young , Cerenius Lage , Söderhäll Kenneth
Published: March 2003

The prophenoloxidase activating enzyme (ppA), a serine proteinase catalyzing the conversion of prophenoloxidase to an active phenoloxidase, has a molecular mass of about 36 kDa in its active form. This protein was cloned from a blood cell cDNA library and its corresponding cDNA of 1736 base pairs encodes a zymogenic protein (proppA) of 468 amino acids. An antibody raised against a synthetic peptide derived from a region of the cDNA sequence could efficiently inhibit the β‐1,3‐glucan triggered activation of prophenoloxidase in vitro. The C‐terminal half of the proppA is composed of a typical serine proteinase domain, with a sequence similar to other invertebrate and vertebrate serine proteinases. The N‐terminal half contains a cationic glycine‐rich domain, a cationic proline‐rich domain and a clip‐domain, in which the disulfide‐bonding pattern is likely to be identical to those of the horseshoe crab big defensin and mammalian β‐defensins. Antibodies made against both the C‐ and the N‐terminal halves recognize two proppAs under reducing conditions. However, under nonreducing conditions only the anti‐C antibody recognized the two proppAs, which suggests that a conformational change takes place upon reduction that allows the anti‐N to react with the N‐terminal half of proppA. The recombinant clip‐domain in crayfish proppA was overexpressed in Escherichia coli and the resulting peptide exhibited antibacterial activity against Gram‐positive bacterial strains such as Micrococcus luteus Ml11 and Bacillus megaterium Bm11 with 50% growth inhibitory concentrations of 1.43 µm and 17.9 µm, respectively. These results suggest that the clip‐domains in proppAs may function as antibacterial peptides.
Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage
Authors: Portaro Fernanda C. V. , Hayashi Mirian A. F. , Silva Celio L. , de Camargo Antonio C. M.
Published: March 2003

The fate of the proteasome‐generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide‐degrading and ‐binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257−264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo‐peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal‐generated oligopeptides in the cytosol.
A change in ionization of the NADP(H)‐binding component (dIII) of proton‐translocating transhydrogenase regulates both hydride transfer and nucleotide release
Authors: Rodrigues Daniel J. , Venning Jamie D. , Quirk Philip G. , Jackson J. Baz
Published: March 2003

Transhydrogenase couples the transfer of hydride‐ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single‐turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped‐flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site‐directed mutagenesis. Two mutants with similar catalytic properties to those of the wild‐type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first‐order rate constants for NADP+ release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)‐binding‐change mechanism. The ionisable group might be a component of the proton‐translocation pathway in the complete enzyme.
Cyclin‐dependent protein kinase 5 (Cdk5) and the regulation of neurofilament metabolism
Authors: Grant Philip , Sharma Pushkar , Pant Harish C.
Published: March 2003

Cyclin‐dependent kinase 5 (Cdk5), a complex of Cdk5 and its activator p35 (Cdk5/p35), phosphorylates diverse substrates which have multifunctional roles in the nervous system. During development, it participates in neuronal differentiation, migration, axon outgrowth and synaptogenesis. Cdk5, acting together with other kinases, phosphorylates numerous KSPXK consensus motifs in diverse cytoskeletal protein target molecules, including neurofilaments, and microtubule associated proteins, tau and MAPs. Phosphorylation regulates the dynamic interactions of cytoskeletal proteins with one another during all aspects of neurogenesis and axon radial growth. In this review we shall focus on Cdk5 and its regulation as it modulates neurofilament metabolism in axon outgrowth, cytoskeletal stabilization and radial growth. We suggest that Cdk5/p35 forms compartmentalized macromolecular complexes of cytoskeletal substrates, other neuronal kinases, phosphatases and activators (‘phosphorylation machines’) which facilitate the dynamic molecular interactions that underlie these processes.
Drug transport by reconstituted P‐glycoprotein in proteoliposomes
Authors: Lu Peihua , Liu Ronghua , Sharom Frances J.
Published: March 2003

The P‐glycoprotein multidrug transporter (Pgp) is an active efflux pump for chemotherapeutic drugs, natural products and hydrophobic peptides. Pgp is envisaged as a ‘hydrophobic vacuum cleaner’, and drugs are believed to gain access to the substrate binding sites from within the membrane, rather than from the aqueous phase. The intimate association of both Pgp and its substrates with the membrane suggests that its function may be regulated by the biophysical properties of the lipid bilayer. Using the high affinity fluorescent substrate tetramethylrosamine (TMR), we have monitored, in real time, transport in proteoliposomes containing reconstituted Pgp. The TMR concentration gradient generated by Pgp was collapsed by the addition of either the ATPase inhibitor, vanadate, or Pgp modulators. TMR transport by Pgp obeyed Michaelis–Menten kinetics with respect to both of its substrates. The Km for ATP was 0.48 mm, close to the Km for ATP hydrolysis, and the Km for TMR was 0.3 µm. TMR transport was inhibited in a concentration‐dependent fashion by verapamil and cyclosporin A, and activated (probably by a positive allosteric effect) by the transport substrate colchicine. TMR transport by Pgp reconstituted into proteoliposomes composed of two synthetic phosphatidylcholines showed a highly unusual biphasic temperature dependence. The rate of TMR transport was relatively high in the rigid gel phase, reached a maximum at the melting temperature of the bilayer, and then decreased in the fluid liquid crystalline phase. This pattern of temperature dependence suggests that the rate of drug transport by Pgp may be dominated by partitioning of drug into the bilayer.
L‐Amino‐acid oxidase from the Malayan pit viper Calloselasma rhodostoma
Authors: Macheroux Peter , Seth Oliver , Bollschweiler Claus , Schwarz Margarete , Kurfürst Manfred , Au Lo‐Chun , Ghisla Sandro
Published: March 2003

Here we report the cDNA‐deduced amino‐acid sequence of l‐amino‐acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD‐dependent ophidian LAAOs to FAD‐dependent oxidases such as monoamine oxidases, d‐amino‐acid oxidases and tryptophan 2‐monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide‐binding fold with monoamine oxidases, tryptophan 2‐monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze‐and pH‐induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl−, prevent pH‐induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.
Generation of human high‐affinity antibodies specific for the fibroblast activation protein by guided selection
Authors: Schmidt Alexej , Müller Dafne , Mersmann Michael , Wüest Thomas , Gerlach Elke , Garin‐Chesa Pilar , Rettig Wolfgang J. , Pfizenmaier Klaus , Moosmayer Dieter
Published: March 2003

Four completely human antibody derivatives [single‐chain‐antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy‐chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding‐competition studies and analysis by fluorescence‐activated cell sorting showed high‐affinity binding (10–20 nm) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable forin vivodiagnostic and tumor‐targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.
Characterization of PknC, a Ser/Thr kinase with broad substrate specificity from the cyanobacterium Anabaena sp. strain PCC 7120
Authors: Gonzalez Leticia , Phalip Vincent , Zhang Cheng‐Cai
Published: March 2003

Eukaryotic‐like protein Ser/Thr and Tyr kinases have only recently been discovered in prokaryotes. In most cases, their biochemical properties have been poorly characterized. The nitrogen‐fixing and heterocyst‐forming cyanobacterium Anabaena sp. strain PCC 7120 houses a family of eukaryotic‐like Ser/Thr kinases. Some of these enzymes are required for cell growth or development under certain conditions. None of them, however, has been shown experimentally to possess Ser/Thr kinase activity. A gene, pknC, encoding a novel putative Ser/Thr kinase was isolated from Anabaena sp. PCC 7120. The recombinant PknC was shown to be phosphorylated on a Thr residue. This phosphorylation was probably due to the autophosphorylation activity of PknC itself because mutation of two amino acid residues within the subdomain II of its catalytic domain eliminated the phosphorylation of PknC. PknC displayed also a Ser kinase activity towards several nonspecific substrates, and the two residues needed for PknC autophosphorylation was equally required for the phosphorylation of other substrates. PknC is thus a Ser/Thr kinase with broad substrate specificity. The activity of PknC is likely to be regulated in vivo in order to limit the spectrum of its substrate specificity.
Peptidomics of the pars intercerebralis–corpus cardiacum complex of the migratory locust, Locusta migratoria
Authors: Clynen Elke , Baggerman Geert , Veelaert Dirk , Cerstiaens Anja , Van der Horst Dick , Harthoorn Lucien , Derua Rita , Waelkens Etienne , De Loof Arnold , Schoofs Liliane
Published: March 2003

The pars intercerebralis–corpora cardiaca system (PI–CC) of insects is the endocrinological equivalent of the hypothalamus–pituitary system of vertebrates. Peptide profiles of the pars intercerebralis and the corpora cardiaca were characterized using simple sampling protocols in combination with MALDI‐TOF and electrospray ionization double quadrupole time of flight (ESI‐Qq‐TOF) mass spectrometric technologies. The results were compared with earlier results of conventional sequencing methods and immunocytochemical methods. In addition to many known peptides, several m/z signals corresponding to putative novel peptides were observed in the corpora cardiaca and/or pars intercerebralis. Furthermore, for a number of peptides evidence was provided about their localization and MALDI‐TOF analysis of the released material from the corpora cardiaca yielded information on the hormonal status of particular brain peptides.
The uptake by cells of 2‐arachidonoylglycerol, an endogenous agonist of cannabinoid receptors
Authors: Bisogno Tiziana , Maccarrone Mauro , De Petrocellis Luciano , Jarrahian Abbas , Finazzi‐Agrò Alessandro , Hillard Cecilia , Di Marzo Vincenzo
Published: March 2003

It is not yet clear if the endocannabinoid 2‐arachidonoylglycerol (2‐AG) is transported into cells through the same membrane transporter mediating the uptake of the other endogenous cannabinoid, anandamide (N‐arachidonoylethanolamine, AEA), and whether this process (a) is regulated by cells and (b) limits 2‐AG pharmacological actions. We have studied simultaneously the facilitated transport of [14C]AEA and [3H]2‐AG into rat C6 glioma cells and found uptake mechanisms with different efficacies but similar affinities for the two compounds (Km 11.0 ± 2.0 and 15.3 ± 3.1 µm, Bmax 1.70 ± 0.30 and 0.24 ± 0.04 nmol·min−1·mg protein−1, respectively). Despite these similar Km values, 2‐AG inhibits [14C]AEA uptake by cells at concentrations (Ki = 30.1 ± 3.9 µm) significantly higher than those required to either 2‐AG or AEA to inhibit [3H]2‐AG uptake (Ki = 18.9 ± 1.8 and 20.5 ± 3.2 µm, respectively). Furthermore: (a) if C6 cells are incubated simultaneously with identical concentrations of [14C]AEA and [3H]2‐AG, only the uptake of the latter compound is significantly decreased as compared to that observed with [3H]2‐AG alone; (b) the uptake of [14C]AEA and [3H]2‐AG by cells is inhibited with the same potency by AM404 (Ki = 7.5 ± 0.7 and 10.2 ± 1.7 µm, respectively) and linvanil (Ki = 9.5 ± 0.7 and 6.4 ± 1.2 µm, respectively), two inhibitors of the AEA membrane transporter; (c) nitric oxide (NO) donors enhance the uptake of both [14C]AEA and [3H]2‐AG, thus suggesting that 2‐AG action can be regulated through NO release; (d) AEA and 2‐AG induce a weak release of NO that can be blocked by a CB1 cannabinoid receptor antagonist, and significantly enhanced in the presence of AM404 and linvanil, thus suggesting that transport into C6 cells limits the action of both endocannabinoids.
Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of α‐glucosidase from Schizosaccharomyces pombe
Authors: Okuyama Masayuki , Okuno Ai , Shimizu Natsumi , Mori Haruhide , Kimura Atsuo , Chiba Seiya
Published: March 2003

cDNA encoding Schizosaccharomyces pombeα‐glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino‐acid residues with Mr 106 138. The deduced amino‐acid sequence showed a high homology to those of α‐glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the α‐glucosidase family II. By site‐directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (‐COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the α‐glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (‐COO−) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.
Hsp90 chaperone complexes are required for the activity and stability of yeast protein kinases Mik1, Wee1 and Swe1
Authors: Goes Fernando S. , Martin Jörg
Published: March 2003

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2‐cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1‐26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function.
Assignment of the disulfide bonds of huwentoxin‐II by Edman degradation sequencing and stepwise thiol modification
Authors: Shu Qin , Huang Renhuai , Liang Songping
Published: March 2003

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin‐II (HWTX‐II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (ΔSer), can be observed in the Cys cycles during Edman degradation of native HWTX‐II. The appearance of two products indicates that the disulfides of HWTX‐II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX‐II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4‐vinylpyridine. Using this method the disulfide bridges of HWTX‐II were assigned as Cys4–Cys18, Cys8–Cys29 and Cys23–Cys34, which is different from that seen in HWTX‐I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n − 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX‐II was confirmed by MALDI‐TOF MS of tryptic fragments of HWTX‐II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX‐II by MALDI‐TOF MS.
Human placental calreticulin
Authors: Højrup Peter , Roepstorff Peter , Houen Gunnar
Published: March 2003

The domain organization and the post‐translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6‐ to 7‐kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N‐terminus were observed, but no stable fragments of intermediate size were found. These results show that the C‐domain of calreticulin is susceptible to proteolytic cleavage and that the N‐ and P‐domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N‐domain, and the third cysteine was found in the reduced form. No post‐translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C‐terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.
The structure of the lipopolysaccharide O‐antigen produced by Flavobacterium psychrophilum (259‐93)
Authors: MacLean Leann L. , Vinogradov Evgeny , Crump Elizabeth M. , Perry Malcolm B. , Kay William W.
Published: March 2003

Flavobacterium psychrophilum, a Gram‐negative bacterium, is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic infections in reared salmonids. In humans Flavobacterium spp. have been associated with neonatal meningitis and septicemia, catheter‐associated bacteremia, and pneumonia. Recently, several F. psychrophilum surface molecules, including lipopolysaccharide (LPS), have been implicated in its pathogenesis and identified as potential vaccine and diagnostic candidate macromolecules. Studies on the LPS produced by the bacterium are reported herein. The structure of the antigenic O‐polysaccharide contained in the LPS of F. psychrophilum was deduced by the application of analytical NMR spectroscopy, mass spectrometry, glycose and methylation analysis, and partial hydrolysis degradations, and was found to be an unbranched polymer of trisaccharide repeating units composed of l‐rhamnose (l‐Rhap), 2‐acetamido‐2‐deoxy‐l‐fucose (l‐FucpNAc) and 2‐acetamido‐4‐((3S,5S)‐3,5‐dihydroxyhexanamido)‐2,4‐dideoxy‐d‐quinovose (d‐Quip2NAc4NR, 2‐N‐acetyl‐4‐N‐((3S,5S)‐3,5‐dihydroxyhexanoyl)‐d‐bacillosamine) (1 : 1 : 1) and having the structure: →4)‐α‐l‐FucpNAc‐(1→3)‐α‐d‐Quip2NAc4NR‐(1→2)‐  α‐l‐Rhap‐(1→ where R is (3S,5S)‐CH3CH(OH)CH2CH(OH)CH2CO‐.
Differential actions of p60c‐Src and Lck kinases on the Ras regulators p120‐GAP and GDP/GTP exchange factor CDC25Mm
Authors: Giglione Carmela , Gonfloni Stefania , Parmeggiani Andrea
Published: March 2003

It is known that the human Ras GTPase activating protein (GAP) p120‐GAP can be phosphorylated by different members of the Src kinase family and recently phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 by proteins of the Src kinase family has been revealed in vivo[Kiyono, M., Kaziro, Y. & Satoh, T. (2000) J. Biol. Chem.275, 5441–5446]. As it still remains unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c‐Src and Lck to phosphorylate these two Ras regulators and have compared the activity of the phosphorylated and unphosphorylated forms. Both kinases were found to phosphorylate full‐length or truncated forms of GAP and GEF. The use of the catalytic domain of p60c‐Src showed that its SH3/SH2 domains are not required for the interaction and the phosphorylation of both regulators. Remarkably, the phosphorylations by the two kinases were accompanied by different functional effects. The phosphorylation of p120‐GAP by p60c‐Src inhibited its ability to stimulate the Ha‐Ras‐GTPase activity, whereas phosphorylation by Lck did not display any effect. A different picture became evident with CDC25Mm; phosphorylation by Lck increased its capacity to stimulate the GDP/GTP exchange on Ha‐Ras, whereas its phosphorylation by p60c‐Src was ineffective. Our results suggest that phosphorylation by p60c‐Src and Lck is a selective process that can modulate the activity of p120‐GAP and CDC25Mm towards Ras proteins.
Design, synthesis and properties of synthetic chlorophyll proteins
Authors: Rau Harald K. , Snigula Heike , Struck Andreas , Robert Bruno , Scheer Hugo , Haehnel Wolfgang
Published: March 2003

A chemoselective method is described for coupling chlorophyll derivatives with an aldehyde group to synthetic peptides or proteins modified with an aminoxyacetyl group at the ε‐amino group of a lysine residue. Three template‐assembled antiparallel four‐helix bundles were synthesized for the ligation of one or two chlorophylls. This was achieved by coupling unprotected peptides to cysteine residues of a cyclic decapeptide by thioether formation. The amphiphilic helices were designed to form a hydrophobic pocket for the chlorophyll derivatives. Chlorophyll derivatives Zn‐methylpheophorbide b and Zn‐methyl‐pyropheophorbide d were used. The aldehyde group of these chlorophyll derivatives was ligated to the modified lysine group to form an oxime bond. The peptide–chlorophyll conjugates were characterized by electrospray mass spectrometry, analytical HPLC, and UV/visible spectroscopy. Two four‐helix bundle chlorophyll conjugates were further characterized by size‐exclusion chromatography, circular dichroism, and resonance Raman spectroscopy.
Generation and epitope mapping of high‐affinity scFv to eukaryotic elongation factor 1A by dual application of phage display
Authors: Kjær Svend , Wind Troels , Ravn Peter , Østergaard Morten , Clark Brian F. C. , Nissim Ahuva
Published: March 2003

To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity‐improve and map epitopes of two previously isolated, low‐affinity single‐chain Fv (scFv) G3 and D1. The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A‐1 and eEF1A‐2. By PCR‐based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage‐based affinity‐selection, two ‘families’ of affinity‐improved scFv were obtained. The scFv of highest affinity, A8, has a Kd of 9 nm to eEF1A‐1. Interestingly, two affinity‐improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv. Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen‐binding. Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413–450 of eEF1A‐1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments. Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage‐antibody repertoire derived scFv were used to select phage‐displayed peptides. Hereby, peptides containing a SFXD motif, matching the SFSD(414–418) sequence found in hEF1A‐1 were isolated. The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold. Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF‐7 fibroblasts. In both cases a perinuclear localization of hEF1A was observed. No evidence for the reported nuclear localization of hEF1A was obtained.
Platelet collagen receptor integrin α2β1activation involves differential participation of ADP‐receptor subtypes P2Y1 and P2Y12 but not intracellular calcium change
Authors: Jung Stephanie M. , Moroi Masaaki
Published: March 2003

In agonist‐induced platelet activation, the collagen platelet receptor integrin α2β1is activated to high‐affinity states through ADP involvement [Jung, S.M. & Moroi, M. (2000)J.Biol.Chem.275, 8016–8026]. Here we determined the ADP‐receptor subtypes involved and their relative contributions to α2β1activation (assessed by soluble‐collagen binding) using the P2Y12 antagonist AR‐C69931MX and P2Y1 antagonists adenosine 3′,5′‐diphosphate (Ado(3,5)PP) and adenosine 3′‐phosphate 5′‐phosphosulfate (AdoPPS). All three inhibited α2β1activation induced by low or high ADP, low thrombin, or low collagen‐related peptide (CRP) concentrations; however, AR‐C69931MX was markedly more inhibitory than the P2Y1 antagonists, suggesting the greater contribution of P2Y12. Inhibition patterns by various combinations of AR‐C69931MX, AdoPPS, and wortmannin suggested that P2Y1 and P2Y12 mediate α2β1activation through different pathways, with possible involvement of phosphoinositide 3‐kinase in both. Low concentrations of the acetoxy‐methyl derivative of 1,2‐bis(o‐aminophenoxy) ethane‐N,N,N′,N′‐tetra‐acetic acid (calcium chelator) markedly decreased α2β1activation by low thrombin or CRP, but did not affect that by low or high ADP. Measurements of intracellular Ca2+level (fluorimetric method) and α2β1activation (soluble‐collagen binding) in the same platelet preparation indicated that α2β1activation via ADP receptors was independent of intracellular Ca2+release. Our data indicate that integrin α2β1activation by ADP occurs through an inside‐out signaling mechanism involving differential contributions by P2Y1 and P2Y12 wherein each contributes to some portion of the activation, with the stronger contribution of P2Y12. Furthermore, intracellular Ca2+increase is not directly related to integrin α2β1activation, meaning that it is separate from the calcium mobilization pathways that these two ADP receptors are involved in.
Purified components of the Escherichia coli Tat protein transport system form a double‐layered ring structure
Authors: Sargent Frank , Gohlke Ulrich , de Leeuw Erik , Stanley Nicola R. , Palmer Tracy , Saibil Helen R. , Berks Ben C.
Published: March 2003

The Escherichia coli twin arginine translocation (Tat) system mediates Sec‐independent export of protein precursors bearing twin arginine signal peptides. The genes tatA, tatB, tatC and tatE code for integral membrane proteins that are components of the Tat pathway. Cells co‐overexpressing tatABCDE show an increased rate of export of a signal peptide‐defective Tat precursor protein and a complex containing the TatA and TatB proteins can be purified from the membranes of such cells. The purified TatAB complex has an apparent molecular mass of 600 kDa as measured by gel permeation chromatography and, like the membranes of wild‐type cells, contains a large molar excess of TatA over TatB. Negative stain electron microscopy of the complex reveals cylindrical structures that may correspond to the Tat protein transport channel.
S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A
Authors: Inoue Akira , Omori Akira , Ichinose Sachiyo , Takahashi Kenichi P. , Kinoshita Yosihiro , Mita Shiro
Published: March 2003

S1 proteins A–D are liberated from thoroughly washed nuclei by mild digestion with DNase I or RNase A, and extracted selectively at pH 4.9 from the reaction supernatants. Here, we characterized the S1 proteins, focusing on protein D2, the most abundant S1 protein in the rat liver, and on protein C2 as well. Using a specific antibody, McAb 351, they were shown to occur in the extranucleolar nucleoplasm, and to be extracted partly in the nuclear soluble fraction. We demonstrate that the S1 proteins in this fraction exist constituting heterogeneous nuclear ribonucleoproteins (hnRNPs), through direct binding to hnRNAs, as revealed by centrifugation on density gradients, immunoprecipitation, and UV cross‐linking. In hnRNPs, protein D2 occurred at nuclease‐hypersensitive sites and C2 in the structures that gave rise to 40 S RNP particles. By microsequencing, protein D2 was identified with a known protein, CArG box motif‐binding factor A (CBF‐A), which has been characterized as a transcriptional repressor, and C2 as its isoform protein. In fact, CBF‐A expressed from its cDNA was indistinguishable from protein D2 in molecular size and immunoreactivity to McAb 351. Thus, the present results demonstrate that S1 proteins C2 and D2 are novel hnRNP proteins, and suggest that the proteins C2 and D2 act in both transcriptional and post‐transcriptional processes in gene expression.
Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralis
Authors: Greller Gerhard , Riek Renate , Boos Winfried
Published: March 2003

We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 °C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein.
CYP11A1 stimulates the hydroxylase activity of CYP11B1 in mitochondria of recombinant yeast in vivo and in vitro
Authors: Cauet Gilles , Balbuena David , Achstetter Tilman , Dumas Bruno
Published: March 2003

In mammals, hydrocortisone synthesis from cholesterol is catalyzed by a set of five specialized enzymes, four of them belonging to the superfamily of cytochrome P‐450 monooxygenases. A recombinant yeast expression system was recently developed for the CYP11B1 (P45011β) enzyme, which performs the 11β hydroxylation of steroids such as 11‐deoxycortisol into hydrocortisone, one of the three mitochondrial cytochrome P‐450 proteins involved in steroidogenesis in mammals. This heterologous system was used to test the potential interaction between CYP11B1 and CYP11A1 (P450scc), the mitochondrial cytochrome P‐450 enzyme responsible for the side chain cleaving of cholesterol. Recombinant CYP11B1 and CYP11A1 were targeted to Saccharomyces cerevisiae mitochondria using the yeast cytochrome oxidase subunit 6 mitochondrial presequence fused to the mature form of the two proteins. In yeast, the presence of CYP11A1 appears to improve 11β hydroxylase activity of CYP11B1 in vivo and in vitro. Fractionation experiments indicate the presence of the two proteins in the same membrane fractions, i.e. inner membrane and contact sites of mitochondria. Thus, yeast mitochondria provide interesting insights to study some molecular and cellular aspects of mammalian steroid synthesis. In particular, recombinant yeast should permit a better understanding of the mechanism permitting the synthesis of steroids (sex steroids, minerallocorticoids and glucocorticoids) with a minimal set of enzymes at physiological level, thus avoiding disease states.
Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin
Authors: Taguchi Goro , Yazawa Teruyoshi , Hayashida Nobuaki , Okazaki Mitsuo
Published: March 2003

Scopoletin is one of the phytoalexins in tobacco. Cells of the T‐13 cell line (Nicotiana tabacumL. Bright Yellow) accumulate a large amount of scopoletin, also known as 7‐hydroxy‐6‐methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1aandNtGT1b) were isolated from a cDNA library derived from the tobacco T‐13 cell line by screening with heterologous cDNAs as a probe. The deduced amino‐acid sequences ofNtGT1aandNtGT1bexhibited 92% identity with each other, ≈ 20–50% identities with other reported glucosyltransferases. Heterologous expression of these genes inEscherichia colishowed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2‐naphthol as a substrate. These recombinant enzymes can utilize UDP‐glucose as the sugar donor, but they can also utilize UDP‐xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.
Enzymatic and electrochemical oxidation of N‐hydroxy compounds
Authors: Xu Feng , Deussen Heinz‐Josef W. , Lopez Brenda , Lam Loi , Li Kaichang
Published: March 2003

A series of N‐hydroxyacetanilide and 1‐hydroxybenzotriazole analogues derivatized by various functional substituents were studied with regard to redox potential, oxidation by laccase, oxidative stability, and correlation to the electronic and steric properties of the substituents. It was found that substituents carrying conjugative/π‐electron function influenced the redox potential more than substituents carrying inductive/σ‐electron function, and that the electron‐transfer from an N‐hydroxy moiety to laccase was significantly affected by the redox potential. Electron‐withdrawing substituents tended to reduce the electron density on the N‐hydroxy group, leading to higher redox potential and lower oxidation rate. Bulky substitution or absence of N‐phenyl tended to increase the Km of the N‐hydroxy group, leading to lower oxidation rate. Oxidized N‐hydroxy compounds were stabilized by N‐phenyl or N‐carbonyl group, but not by N‐azo or highly strained structure. Potential implication of these effects on laccase‐based, N‐hydroxy compound‐mediated biocatalysis is discussed.
Highly efficient targeting and accumulation of a Fabfragment within the secretory pathway and apoplast ofArabidopsis thaliana
Authors: Peeters Koen , De Wilde Chris , Depicker Ann
Published: March 2003

To further improve antibody production in plants, constructs were designed to minimize transgene silencing and to retain a Fabfragment within the secretory pathway of transgenicArabidopsis thalianaplants. The levels of antibody accumulation suggest that placing the sequences that encode Fd and light chain under the control of nonidentical 3′ regions reduces susceptibility to post‐transcriptional gene silencing compared with when the individual polypeptide‐encoding sequences are placed under the control of identical 3′ regions. High levels of accumulation (up to 6% of total soluble protein) were found for both secreted and intracellularly targeted antibody fragments. Immunofluorescence microscopic analysis showed that Fabfragments devoid of any additional C‐terminal sequence were efficiently secreted, whereas retention of Fabfragments within the endomembrane system of the secretory pathway was achieved by C‐terminal fusion of the DIKDEL sequence to the antibody light chain. Furthermore, analysis by immunoprecipitation and ELISA showed that intracellular retention of antibody fragments did not affect antigen‐binding activity, and more than 80% of the isolated antibody fragments were found to bind antigen. Taken together, our results provide improvements to the technology of recombinant antibody production in transgenic plants.
Stepwise proteolytic removal of the β subdomain in α‐lactalbumin
Authors: Polverino de Laureto Patrizia , Vinante Daniela , Scaramella Elena , Frare Erica , Fontana Angelo
Published: March 2003

Bovine α‐lactalbumin (α‐LA) is an α/β protein which adopts partly folded states when dissolved at low pH (A‐state), by removal of the protein‐bound calcium at neutral pH and low salt concentration (apo‐state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A‐state of α‐LA at pH 2.0, considered a prototype molten globule, has a native‐like fold in which the helical core is mostly retained, while the β subdomain is less structured. Here, we investigate the conformational features of three derivatives of α‐LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino‐acid residues of the β subdomain of the native protein (approximately from residue 34 to 57). These α‐LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked α‐LA species consisting of fragments 1‐,3–40 and 41–123 (nicked‐LA) was prepared by thermolytic digestion of the 123‐residue chain of α‐LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1–40 and 53–123 (desβ1‐LA) or fragments 1–34 and 54‐,57–123 (desβ2‐LA) were obtained by digestion of α‐LA with pepsin in acid or with proteinase K at neutral pH in its apo‐state, respectively. The two protein fragments of nicked or gapped α‐LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact α‐LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far‐ and near‐UV region reveal a much more labile tertiary structure in the α‐LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three α‐LA variants adopt a conformational state essentially identical to the molten globule displayed by intact α‐LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8‐anilinonaphthalene‐1‐sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the β subdomain can be removed from the α‐LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the α‐LA molten globule only the α domain is structured.
Oxidized low density lipoproteins downregulate LPS‐induced platelet‐activating factor receptor expression in human monocyte‐derived macrophages
Authors: Hourton Delphine , Stengel Dominique , Chapman M. John , Ninio Ewa
Published: March 2003

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet‐activating‐factor (PAF). We investigated the effect of oxidized low‐density lipoprotein (oxLDL) on lipopolysaccharide (LPS)‐induced PAF receptor (PAF‐R) expression in human macrophages and the implication of the nuclear factor (NF)‐κB in this regulation. LPS‐treatment (1 µg·mL−1) of macrophages increased PAF binding and PAF‐R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low‐density lipoprotein [ox24hLDL; 100 µg·mL−1; thiobarbituric acid reacting substances: 31 ± 3 nmol equiv. malondialdehyde (MDA)·mg protein LDL−1] diminished PAF‐R expression (−69%; P < 0.05) and mRNA level (− 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF‐κB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS‐induced binding of NF‐κB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 µm), a major component of oxLDL, inhibited the LPS‐induced NF‐κB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF‐R expression in human macrophages by inhibiting LPS‐induced NF‐κB binding to DNA.
Escherichia coli RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α2β subassembly
Authors: Ghosh Pallavi , Ishihama Akira , Chatterji Dipankar
Published: March 2003

The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω‐less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X‐ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N‐terminus and C‐terminus. We show here that ω binds only the intact β′ subunit and not the β′ N‐terminal domain or β′ C‐terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8‐protease‐resistant 52‐amino‐acid‐long N‐terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N‐terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly.
Irreversible inhibition of pig kidney copper‐containing amine oxidase by sodium and lithium ions
Authors: Padiglia Alessandra , Medda Rosaria , Lorrai Anita , Paci Maurizio , Pedersen Jens Z. , Boffi Alberto , Bellelli Andrea , Agrò Alessandro Finazzi , Floris Giovanni
Published: March 2003

Copper amine oxidase was found to be inhibited in a complex way by small alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source‐specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co‐ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.