bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of recombinant Arabidopsis thaliana threonine synthase
Authors: Laber Bernd , Maurer Wolfgang , Hanke Christiane , Gräfe Susanna , Ehlert Stephan , Messerschmidt Albrecht , Clausen Tim
Published: March 2003

Threonine synthase (TS) catalyses the last step in the biosynthesis of threonine, the pyridoxal 5′‐phosphate dependent conversion of l‐homoserine phosphate (HSerP) into l‐threonine and inorganic phosphate. Recombinant Arabidopsis thaliana TS (aTS) was characterized to compare a higher plant TS with its counterparts from Escherichia coli and yeast. This comparison revealed several unique properties of aTS: (a) aTS is a regulatory enzyme whose activity was increased up to 85‐fold by S‐adenosyl‐l‐methionine (SAM) and specifically inhibited by AMP; (b) HSerP analogues shown previously to be potent inhibitors of E. coli TS failed to inhibit aTS; and (c) aTS was a dimer, while the E. coli and yeast enzymes are monomers. The N‐terminal region of aTS is essential for its regulatory properties and protects against inhibition by HSerP analogues, as an aTS devoid of 77 N‐terminal residues was neither activated by SAM nor inhibited by AMP, but was inhibited by HSerP analogues. The C‐terminal region of aTS seems to be involved in dimer formation, as the N‐terminally truncated aTS was also found to be a dimer. These conclusions are supported by a multiple amino‐acid sequence alignment, which revealed the existence of two TS subfamilies. aTS was classified as a member of subfamily 1 and its N‐terminus is at least 35 residues longer than those of any nonplant TS. Monomeric E. coli and yeast TS are members of subfamily 2, characterized by C‐termini extending about 50 residues over those of subfamily 1 members. As a first step towards a better understanding of the properties of aTS, the enzyme was crystallized by the sitting drop vapour diffusion method. The crystals diffracted to beyond 0.28 nm resolution and belonged to the space group P222 (unit cell parameters: a = 6.16 nm, b = 10.54 nm, c = 14.63 nm, α = β = γ = 90°).
N‐Tail translocation in a eukaryotic polytopic membrane protein
Authors: Monné Magnus , Gafvelin Guro , Nilsson Robert , von Heijne Gunnar
Published: March 2003

We have used the natural N‐glycosylation site in the N‐tail of cig30, a eukaryotic polytopic membrane protein, as a marker for N‐tail translocation across the microsomal membrane. Analysis of C‐terminally truncated cig30 constructs reveals that the first transmembrane segment is sufficient for translocation of the wild‐type N‐tail; in contrast, in a mutant with four arginines introduced into the N‐tail the second transmembrane segment is also required for efficient N‐tail translocation. Our observations imply a non‐sequential assembly mechanism in which the ultimate location of the N‐tail relative to the membrane may depend on more than one transmembrane segment.
The metaphase to anaphase transition
Authors: Farr Katie A. , Cohen‐Fix Orna
Published: March 2003

The metaphase to anaphase transition is a point of no return; the duplicated sister chromatids segregate to the future daughter cells, and any mistake in this process may be deleterious to both progeny. At the heart of this process lies the anaphase inhibitor, which must be degraded in order for this transition to take place. The degradation of the anaphase inhibitor occurs via the ubiquitin‐degradation pathway, and it involves the activity of the cyclosome/anaphase promoting complex (APC). The fidelity of the metaphase to anaphase transition is ensured by several different regulatory mechanisms that modulate the activity of the cyclosome/APC. Great advancements have been made in this field in the past few years, but many questions still remain to be answered.
p‐Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var. thunbergii
Authors: Akiyama Takumi , Shibuya Masaaki , Liu Hong‐Min , Ebizuka Yutaka
Published: March 2003

Chalcone synthase and stilbene synthase are plant‐specific polyketide synthases. They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions. They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase. Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var. thunbergii. They were expressed in Escherichia coli in order to determine their enzyme functions. Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene. However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein. This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.
A new member of the α4‐related molecule (α4‐b) that binds to the protein phosphatase 2A is expressed selectively in the brain and testis
Authors: Maeda Kazuhiko , Inui Seiji , Tanaka Hideaki , Sakaguchi Nobuo
Published: March 2003

A murine α4, identified in lymphocytes, binds to protein phosphatase 2A (PP2A). We found another murine α4‐related gene (named α4‐b) expressed selectively in the brain and testis. The α4‐b transcript is expressed in the brain and testis, but is not detected in the spleen, thymus, bone marrow, liver, kidney, lung, heart or muscle. In‐situ RNA hybridization analysis suggested that α4‐b is expressed in most neuronal cells in the brain, but it is not expressed in the glial cells. The α4‐b cDNA encodes a putative protein that is highly homologous (66% identity in amino‐acid sequence) to the α4 molecule. The α4‐b protein associates with the catalytic subunit of PP2A (PP2Ac), suggesting that the α4‐b protein is involved in the regulation of phosphatase activity in neuronal cells.
Transferrins, the mechanism of iron release by ovotransferrin
Authors: Abdallah Fadi Bou , Chahine Jean‐Michel El Hage
Published: March 2003

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (≤ 5 ms) decarbonation of the iron‐loaded protein. Iron release from both sites of the protein is controlled by what appear to be slow proton transfers. The N‐site loses its iron first in two steps, the first occurring in the tenth of a second range with second order rate constant k1 = (2.30 ± 0.10) × 104 m−1·s−1, first order rate constant k−1 = (1.40 ± 0.10) s−1 and equilibrium constant K1a = (60 ± 6) µm. The second step occurs in the second range with a second order rate constant k2 = (5.2 ± 0.15) × 103 m−1·s−1, first order rate constant k−2 = (0.2 ± 0.02) s−1 and equilibrium constant K2a = (39 ± 5) µm. Iron is afterward lost from the C‐site of the protein by two different pathways, one in the presence of a strong Fe(III) ligand such as citrate and the other in the presence of weak ligands such as formate or acetate. The first step, common to both paths, is a slow proton uptake which occurs in the tens of second range with a second order rate constant k3 = (1.22 ± 0.03) × 103 m−1·s−1 and equilibrium constant K3a = (1.0 ± 0.1) mm. In the presence of citrate, this step is followed by formation of an intermediate complex with monoferric ovotransferrin; stability constant KLC = (0.435 ± 0.015) mm. This last step is rate‐controlled by slow proton gain which occurs in the hundred second range with a second order rate constant k4 = (1.05 ± 0.05) × 104 m−1·s−1, first order rate constant k−4 = (1.0 ± 0.1) × 10−2 s−1 and equilibrium constant K4a = (0.95 ± 0.15) µm. In the presence of a weak iron(III) ligand such as acetate or formate, formation of an intermediate complex is not detected and iron release is controlled by two final slow proton uptakes. The first occurs in the hundred to thousand second range, second order rate constant k5 = (6.90 ± 0.30) × 106 m−1·s−1. The last step occurs in the thousand second range. Iron release by ovotransferrin is similar but not identical to that of serum‐transferrin. It is slower and occurs at lower pH values. However, as seen for serum‐transferrin, it seems to involve the protonation of the amino acid side‐chains involved in iron co‐ordination and perhaps those implicated in interdomain H‐bonds. The observed proton transfers are, then, probably controlled by the change in conformation of the binding lobes from closed when iron‐loaded to open in the apo‐form.
Induction of ferritin and heat shock proteins by prostaglandin A1 in human monocytes
Authors: Elia Giuliano , Polla Barbara , Rossi Antonio , Santoro M. Gabriella
Published: March 2003

Prostaglandins of the A type (PGA) exert a cytoprotective activity during hyperthermia and virus infection. This effect is associated with induction of heat shock proteins (HSP) in mammalian cells. We now report that, in human monocytes, PGA1 is able to induce the synthesis of the iron‐binding, redox‐regulated protein ferritin. L‐chain ferritin induction is consequent to a substantial increase in the accumulation of L‐chain ferritin transcripts in PGA1‐treated cells, whereas H‐chain ferritin is regulated post‐transcriptionally, consequently to reduction of iron‐regulatory protein binding to iron‐responsive elements in ferritin mRNA. Ferritin induction is specific for cyclopentenone prostaglandins (PGA1, PGA2, PGJ2, Δ12‐PGJ2), whereas other arachidonic acid (AA) metabolites have no effect. In human monocytes, PGA1 also induces heat shock gene transcription via heat shock factor activation, as well as the synthesis of the oxidative‐stress protein heme oxygenase (HOS). Differently from HSP, the induction of ferritin by PGA1 is specific for monocytes. Monocytes/macrophages play a pivotal role in inflammation, controlling iron metabolism and releasing a variety of mediators, including proinflammatory reactive oxygen species (ROS), cytokines and AA metabolites. As ferritin, together with hsp70 and HO, plays a key role in protection from oxidant damage, these results suggest that PGA1 may have cytoprotective activity also during oxidative injury.
Scorpion toxins specific for Na+‐channels
Authors: Possani Lourival D. , Becerril Baltazar , Delepierre Muriel , Tytgat Jan
Published: March 2003

Na+‐channel specific scorpion toxins are peptides of 60–76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three‐dimensional structure has been solved by X‐ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of α‐helix plus a triple‐stranded β‐sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+‐channel function, affecting either the inactivation (α‐toxins) or the activation (β‐toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical α‐ and β‐types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+‐channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or ‘active sites’ of these peptides with Na+‐channel functions. Segments containing positively charged residues in special locations, such as the five‐residue turn, the turn between the second and the third β‐strands, the C‐terminal residues and a segment of the N‐terminal region from residues 2–11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well‐folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.
Lumichrome
Authors: Tsukamoto Sachiko , Kato Haruko , Hirota Hiroshi , Fusetani Nobuhiro
Published: March 2003

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval‐conditioned seawater. However, such a natural ‘inducer’ has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis‐inducing substance from the larvae, the larval‐conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species‐specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis‐inducing substance to be identified in ascidians.
Co‐operative binding sites for transported substrates in the multiple drug resistance transporter Mdr1
Authors: Buxbaum Engelbert
Published: March 2003

Suramin, a known inhibitor of ATP binding enzymes with six negatively charged sulfonic acid groups, stimulated the ATPase activity of the multiple drug resistance transporter Mdr1 in low concentrations by acting as a substrate and by increasing the affinity for both verapamil and ATP. At higher concentrations suramin inhibited the ATPase activity competitively with respect to ATP and noncompetitively with respect to verapamil. This indicates an interaction of suramin with the ATP site. Verapamil itself activated the ATPase activity of Mdr1 only at moderate concentrations, but showed substrate inhibition at higher concentrations. This was also observed for progesterone, which decreased the Ki of Mdr1 for verapamil but increased the Km. Additionally, verapamil increased the Hill coefficient of Mdr1 for progesterone from 1.1 to 3.2. These results indicate the existence of multiple binding sites (at least four for progesterone) for transported substrate in Mdr1 and a complicated mode of interactions between them.
The crystal structure of a wheat nonspecific lipid transfer protein (ns‐LTP1) complexed with two molecules of phospholipid at 2.1 Å resolution
Authors: Charvolin Delphine , Douliez Jean‐Paul , Marion Didier , Cohen‐Addad Claudine , Pebay‐Peyroula Eva
Published: March 2003

Nonspecific lipid transfer proteins (ns‐LTP1) form a multigenic protein family in plants. In vitro they are able to bind all sort of lipids but their function, in vivo, remains speculative. A ns‐LTP1 isolated from wheat seed was crystallized in the presence of lyso‐myristoyl‐phosphatidylcholine (LMPC). The structure was solved by molecular replacement and refined to 2.1 Å resolution to an R‐factor of 16.3% and a free R‐factor of 21.3%. It reveals for the first time that the protein binds two LMPC molecules that are inserted head to tail in a hydrophobic cavity. A detailed study of the structure leads to the conclusion that there are two lipid‐binding sites, one of which shows a higher affinity for the LMPC than the other. Comparison with other structures of lipid‐bound ns‐LTP1 suggests that the presence of two binding sites is a general feature of plant ns‐LTP1.
Cysteine biosynthesis inChlamydomonas reinhardtii
Authors: Ravina Cristina G. , Barroso Consuelo , Vega José M. , Gotor Cecilia
Published: March 2003

A cDNA,Cys1ACr, encoding an isoform ofO‐acetylserine(thiol) lyase has been isolated fromChlamydomonas reinhardtii, using a PCR‐based approach. The inclusion of dimethylsulfoxide in the PCR reaction has been demonstrated to be essential for the correct amplification ofC. reinhardtiitemplates with complex secondary structures caused by a high G + C content. The deduced amino acid sequence exhibited highest similarity with plantO‐acetylserine(thiol)lyase isoforms, indicating that theC. reinhardtiienzyme was structurally more similar to higher plantO‐acetylserine(thiol)lyase than to the corresponding prokaryotic enzymes. The N‐terminal extension present in Cys1ACr showed several characteristics of an organellar transit peptide, with a length typical forC. reinhardtii. Southern blot analysis suggested that theC. reinhardtiigenome may contain a single copy of the organellarO‐acetylserine(thiol)lyase gene.O‐acetylserine(thiol)lyase activity was strongly induced by sulfur‐deficient conditions (up to sevenfold the level observed in a sulfur‐repleted cell culture) and required the presence of a nitrogen source. Northern blot analysis showed a different pattern of regulation ofCys1ACrto that observed at the activity level. To obtain an increase of transcript abundance a longer period of sulfur limitation was required, reaching a maximum level of ≈ threefoldCys1ACrmRNA when compared with the level of a sulfate‐grown culture.
Microtubule‐binding property of microtubule‐associated protein 2 differs from that of microtubule‐associated protein 4 and tau
Authors: Tokuraku Kiyotaka , Katsuki Miho , Matui Takanori , Kuroya Tomomi , Kotani Susumu
Published: March 2003

The microtubule‐binding domains of microtubule‐associated protein (MAP) 2, MAP4, and tau are structurally similar [Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem.265, 13849–13855]. To compare the microtubule‐binding mechanisms of the three MAPs, we performed a quantitative competition analysis using the three MAPs and the microtubule‐binding domain fragment of MAP4 (PA4T fragment). The two‐cycled microtubule protein fraction from bovine brain contains MAP1, MAP2, MAP4, and tau. When an excess of the PA4T fragment was added to the microtubule protein fraction, MAP4 and tau were completely released from the microtubules, while MAP1 remained bound. MAP2 was only partially released from the microtubules. The competition between MAP2 and MAP4 was further analyzed using purified MAP2, the PA4T fragment, and tubulin. About half of the MAP2 was still bound to the microtubules, even in the presence of an excess amount of the PA4T fragment. The microtubule‐binding mechanisms of MAP2 and MAP4 seem to be different, in spite of their similar primary structures.
Slow unfolding and refolding kinetics of the mesophilic Rop wild‐type protein in the transition range
Authors: Rosengarth Anja , Rösgen Jörg , Hinz Hans‐Jürgen
Published: March 2003

We describe the guanidinium hydrochloride induced folding kinetics of the four‐helix‐bundle protein Rop wild‐type (wt) under equilibrium conditions at three temperatures. The choice of appropriate denaturant conditions inside the transition range permitted, in combination with equilibrium transition curves, the determination of both unfolding and refolding rate constants. The ratio of the rate constants at zero denaturant concentration provided equilibrium constants and standard free energy changes that are in good agreement with values obtained in previous differential scanning calorimetry studies. The ΔG0D values for 19, 25 and 40 °C calculated from the present kinetic studies are, respectively, 66.8, 70.8 and 57.2 kJ·mol−1. The unfolding reactions are extremely slow under these conditions. Equilibrium was reached only after 18, 12 and 6 days at 19, 25 and 40 °C. These results demonstrate that for Rop wt high stability correlates with slow folding kinetics.
Different protein turnover of interleukin‐6‐type cytokine signalling components
Authors: Siewert Elmar , Müller‐Esterl Werner , Starr Robin , Heinrich Peter C. , Schaper Fred
Published: March 2003

Interleukin (IL)‐6 and IL‐6‐type cytokines signal through the gp130/Jak/STAT signal transduction pathway. The key components involved are the signal transducing receptor subunit gp130, the Janus kinases Jak1, Jak2 and Tyk2, STAT1 and STAT3 of the family of signal transducers and activators of transcription, the protein tyrosine phosphatase SHP2 and the suppressors of cytokine signalling SOCS1, SOCS2 and SOCS3. Whereas considerable information has been accumulated concerning the time‐course of activation for the individual signalling molecules, data on the availability of the proteins involved in IL‐6‐type cytokine signal transduction are scarce. Nevertheless, availability of these molecules, determined by the balance of protein synthesis and degradation, also influences IL‐6‐type cytokine signal transduction. Here, we present a comprehensive set of data on the half‐lives of the key molecules involved in the IL‐6 signal transduction pathway. The turnover rates for the various proteins differ substantially. Three groups of signalling proteins can be discriminated: whereas the feedback inhibitors SOCS1, SOCS2 and SOCS3 are very short‐lived, STAT1, STAT3 and SHP2 have an extremely slow turnover rate. Interestingly, the half‐life of STAT3β, a splice variant of STAT3α, is reduced to almost 50% of the half‐life of STAT3α. The Janus kinases Jak1, Jak2, Tyk2 and gp130 show intermediate half‐lives. Our data imply that signalling components activated by post‐translational modifications are long‐lived whereas the activity of very short‐lived proteins is regulated mainly at the transcriptional level.
The activity of the human interleukin‐5 conserved lymphokine element 0 is regulated by octamer factors in human cells
Authors: Thomas Marc A. , Mordvinov Viatcheslav A. , Sanderson Colin J.
Published: March 2003

Interleukin‐5 (IL‐5) controls the development of eosinophilia and contributes to a number of disease states including asthma. Expression of IL‐5 is inducible under tight transcriptional regulation. This requires the contribution of several promoter elements; however, the conserved lymphokine element 0 (CLE0) in particular, is essential for expression of IL‐5. In this study, we report the nuclear factors which regulate human IL‐5 CLE0 activity in the human cell line PER‐117. Using specific antibodies, we identified the transcriptional factors Oct‐1 and Oct‐2 binding to the 5′ region of the CLE0 element. The involvement of Oct factors with CLE0 has not been reported previously in any of the lymphokines. In addition, the CLE0 element also appeared to complex with the transcriptional activator AP‐1, consisting of the family members Jun D and Fra‐2. We observed the binding of Oct‐1 to be constitutive in comparison to Oct‐2 and AP‐1, both of which were induced in response to cell activation by PMA/A23187. Although the interaction of all three factors with CLE0 was closely linked and overlapping, residues critical to their binding were identified. We demonstrate, using site‐directed mutagenesis and cotransfection experiments, that the CLE0 element is indispensable for IL‐5 promoter activity and that Octamer factors contribute to the positive regulation of the hIL‐5 gene.
Biosynthesis and immunobiochemical characterization of gp17/GCDFP‐15
Authors: Caputo Emilia , Carratore Vitale , Ciullo Marina , Tiberio Claudia , Mani Jean‐Claude , Piatier‐Tonneau Dominique , Guardiola John
Published: March 2003

The gp17 factor is a secretory product of human seminal vesicle cells which binds to CD4 and acts as a potent inhibitor of T lymphocyte apoptosis induced by CD4 crosslinking and subsequent T‐cell receptor (TCR) activation. The protein is identical to gross cystic disease fluid protein‐15 (GCDFP‐15), a breast tumor secretory marker PIP (prolactin inducible protein), a prolactin‐controlled and androgen‐controlled protein; secretory actin binding protein (SABP), a seminal plasma actin binding protein and extra‐parotid glycoprotein (EP‐GP), a secretory protein from the salivary gland. The structure of this protein has not yet been elucidated and no biological function has been clearly attributed to date. Expression of recombinant gp17/GCDFP‐15 cDNA in bacteria and insect cells leads to the production of a misfolded insoluble protein. In this study, we describe the production of gp17/GCDFP‐15 in two different eukaryotic systems, namely HeLa cells and the Pichia pastoris yeast. Using constructs in which gp17/GCDFP‐15 was tagged with enhanced green fluorescent protein (EGFP) in various combinations, we observed expression only when the fusion protein was directed to the secretory compartment by the correct signal peptide. The resulting fluorescent protein was inefficiently secreted, thus suggesting that gp17/GCDFP‐15 is not appropriately post‐translationally processed and/or transported in HeLa cells. The use of the P. pastoris secretory pathway allowed instead the accumulation in the culture medium of a GCDFP‐15/gp17 species which retained the ability to bind to CD4 and also most of the biochemical and immunological properties of the native protein. The production of an active recombinant molecule opens the way to correlate the structural properties of this peculiar factor to its ability to bind several proteins, including CD4, and to block CD4‐mediated T cell programmed death.
Structural versatility of bovine ribonuclease A
Authors: Gotte Giovanni , Bertoldi Mariarita , Libonati Massimo
Published: March 2003

Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII‐A) from 40% acetic acid solutions leads to the formation of ≈ 14 aggregated species that can be separated by ion‐exchange chromatography. Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously [Gotte, G. & Libonati, M. (1998) Two different forms of aggregated dimers of ribonuclease A. Biochim. Biophys. Acta1386, 106–112]. We also have possible evidence for the existence of two forms of pentameric RNase A. The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion‐exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions. Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues. All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A)·poly(U), viral double‐stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species. On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A.
Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation
Authors: Juarranz Maria G. , Van Rampelbergh Jean , Gourlet Philippe , De Neef Phillippe , Cnudde Johnny , Robberecht Patrick , Waelbroeck Magali
Published: March 2003

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1–7)/GRF (8–27) and VIP1 antagonist, Ac His1 [D‐Phe2, Lys15, Arg16, Leu27] VIP (3–7)/GRF (8–27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N‐terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N‐terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.
Structure of the Alzheimer β‐amyloid peptide (25–35) and its interaction with negatively charged phospholipid vesicles
Authors: del Mar Martínez‐Senac María , Villalaín José , Gómez‐Fernández Juan C.
Published: March 2003

The secondary structure of amyloid βAP(25–35) peptide was studied in pure form and in the presence of different phospholipid vesicles, by using Fourier transform infrared spectroscopy (FT‐IR). Pure peptide aggregated with time, forming fibrils with β‐structure. Phospholipid vesicles formed by negatively charged phospholipids such as 1,2‐dimyristoyl‐sn‐glycerol‐3‐phospho‐l‐serine (Myr2PtdSer), 1,2‐dimyristoyl‐sn‐glycerol‐3‐phospho‐rac‐1‐glycerol (Myr2PtdGro) and 1,2‐dimyristoyl‐sn‐glycerol 3‐phosphate (Myr2PtdH), greatly accelerated the aggregation of the peptide. However, the presence of vesicles formed by the zwitterionic phospholipid, 1,2‐dimyristoyl‐sn‐glycerol‐3‐phosphocholine (Myr2PtdCho), slowed down the aggregation process. Differential scanning calorimetry (DSC) measurements showed that the effect of βAP(25–35) on the gel to crystal liquid phase transition was small at neutral pH for negatively charged phospholipids and practically nil for Myr2PtdCho. In the case of Myr2PtdSer the effect was also zero at pH 9 but the effect was large at pH 3. The effect on Myr2PtdH was not, however, very dependent on pH. These results were fully confirmed by the observation through FT‐IR of the change with temperature of the CH2 antisymmetric stretching vibration. The case of Myr2PtdGro was special as this phospholipid presents polymorphism giving solid quasicrystalline phases when it is not sufficiently hydrated, and it is remarkable that βAP(25–35) was able to induce the formation of crystalline phases in samples prepared through a method which ensure a good hydration of phospholipid. These results show that the interaction of amyloid βAP(25–35) peptide with phospholipids is based on electrostatic interactions, that these interactions favour the aggregation of the peptides, and that the presence of the aggregates may disturb the lipid‐water interphase of the membrane.
Molten globule‐like state of human serum albumin at low pH
Authors: Muzammil Salman , Kumar Yogesh , Tayyab Saad
Published: March 2003

Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid‐denatured E form using far‐UV and near‐UV CD, binding of a hydrophobic probe, 1‐anilinonaphthalene‐8‐sulfonic acid (ANS), thermal transition by far‐UV and near‐UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far‐UV CD spectra. The near‐UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non‐polar clusters. The temperature‐dependence of both near‐UV and far‐UV CD signals did not exhibit a co‐operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid‐denatured state, it is buried in the interior in a non‐polar environment. Intrinsic viscosity measurements showed that the acid‐denatured state is relatively compact compared with that of the denatured state in 7 m guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.
Structural studies of the O‐specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide
Authors: Jachymek Wojciech , Czaja Jolanta , Niedziela Tomasz , Lugowski Czeslaw , Kenne Lennart
Published: March 2003

The structure of the O‐specific side‐chain of the Hafnia alvei strain PCM 1207 lipopolysaccharide (LPS) has been investigated. Methylation analysis, partial acid hydrolysis, matrix‐assisted laser‐desorption ionization time‐of‐flight (MALDI‐TOF) MS, fast atom bombardment (FAB)‐MS/MS and 1H‐ and 13C‐NMR spectroscopy were the principal methods used. Glycerol phosphate was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established:

The polysaccharide is partially (≈ 10%) substituted with O‐acetyl groups. The lipopolysaccharide was also subjected to high resolution magic angle spinning (HR‐MAS) NMR analysis, which showed both the signals of the O‐specific polysaccharide as well as several signals from unsubstituted core oligosaccharides. This confirmed the presence of the described structure in the native LPS.
Functional protective role for mucin glycosylated repetitive domains
Authors: Loomes Kerry M. , Senior Hugh E. , West Paula M. , Roberton Anthony M.
Published: March 2003

Mucins carry out a number of protective roles, some of which are more easily studied than others. One mucin function is believed to be the protection of the mucosal epithelium against acidic and proteolytic damage in the stomach and intestines. In the present work, a portion of stomach mucin tandem repeat sequence (Muc6) was joined to the catalytic domain of a reporter enzyme [human milk cholesterol esterase (CE)] to determine whether the former can protect the latter protein from damage. This Muc6 domain replaced a unique series of glycosylated C‐terminal repeats normally present in CE. The chimeric protein (CE/Muc6) was expressed in two different cell lines and its properties compared to recombinant full‐length CE and a truncated version of CE which contained only the catalytic domain (CE/trunc). Results showed that both CE and CE/Muc6 were resistant to denaturation by acid and to proteolysis by pepsin at low pH values or by pancreatic proteases compared to CE/trunc. Thus, a stomach Muc6 domain is sufficient to confer stability on the CE catalytic domain, demonstrating a protective effect by a glycosylated mucin sequence.
Purification and characterization of thyroid‐hormone‐binding protein from masu salmon serum
Authors: Yamauchi Kiyoshi , Nakajima Jun‐ichiro , Hayashi Hiroaki , Hara Akihiko , Yamauchi Kohei
Published: March 2003

We purified a thyroid‐hormone‐binding protein (THBP) from serum of masu salmon at the stage of smoltification when the concentrations of endogenous thyroid hormones in plasma reach the highest levels. All steps of sequential column chromatography suggest that this THBP is responsible for most l‐3,5,3′‐triiodothyronine‐binding activity in serum at this stage. The molecular mass of this protein was estimated to be 60 kDa by gel filtration but only 15 kDa by SDS/PAGE, which suggests that it is comprised of four identical subunits. The amino acid sequence of its N‐terminal portion was highly similar to those of vertebrate transthyretins. These molecular features indicate that masu salmon THBP is a homolog of transthyretins from tetrapods. However, in contrast with mammalian transthyretins, the affinity of masu salmon transthyretin for l‐3,5,3′‐triiodothyronine was three times greater than for l‐thyroxine. This rank order affinity is similar to that of avian and frog transthyretins. Scatchard analysis revealed that masu salmon transthyretin possesses a single class of binding site for l‐3,5,3′‐triiodothyronine, with a Kd of 13.8 nm at 0 °C. Taken together with the data reported by Chang et al. [Eur. J. Biochem. (1999) 259, 534–542], these results suggest that transthyretin has changed from a l‐3,5,3′‐triiodothyronine‐carrier protein to a l‐thyroxine‐carrier protein during mammalian evolution.
Epitope analysis and molecular modeling reveal the topography of the C‐terminal peptide of the β‐subunit of human chorionic gonadotropin
Authors: Venkatesh Natarajan , Krishnaswamy Sankaran , Meuris Sylvain , Murthy Gundulpet Satyanarayana
Published: March 2003

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common α‐subunit and a hormone‐specific β‐subunit. Among the gonadotropin β‐subunits, greater than 85% homology exists between lutropin (hLH)β and hCGβ in their first 114 amino acid residues. However, unlike hLHβ, hCGβ contains a 31‐amino acid hydrophilic stretch at its carboxyl end (CTPβ: C‐terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPβ remains unknown. In this study, we have attempted to define the topology of CTPβ using mAb probes. We investigated three epitopes on hCGα, which are hidden in the hCGαβ dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPβ. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPβ. Consistent with this view, several treatments of hCG that removed CTPβ unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPβ on the α‐subunit, we used an epitope‐mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods202, 173–182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPβ. Further, we were also able to build a model of CTPβ, completely independently of the epitope‐mapping studies, using a homology‐based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci.4 (Suppl. 2), 86–97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPβ are in contact with hCGα in the native hCG dimer.
Isolation and characterization of hydrophobic polypeptides in human bile
Authors: Stark Margareta , Jörnvall Hans , Johansson Jan
Published: March 2003

Polypeptides were isolated from human bile by extraction with chloroform/methanol, followed by reversed‐phase chromatography in methanol/ethylene chloride and gel filtration in chloroform/methanol. Peptides were characterized by SDS/PAGE, sequence analysis and matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry. This identified haemoglobin α chain, ATP synthase lipid‐binding protein subunit 9, an N‐terminal fragment of mac25/insulin‐like growth factor‐binding protein 7 and an internal fragment of monocyte differentiation antigen CD14, all not described previously in bile. In addition, α1‐antitrypsin, known in bile from previous work, was also identified. The hydrophobic character of haemoglobin α chain is not apparent from its amino acid sequence, but the other polypeptides all have major hydrophobic segments. These results show that several proteins are removed upon organic solvent extraction used for delipidation during the preparation of samples for proteome analysis. Several of the polypeptides found are unexpectedly present in bile, suggesting that specific excretion mechanisms may be involved.
Internal regulation of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes
Authors: Ainscow Edward K. , Brand Martin D.
Published: March 2003

Previously [Ainscow, E.K. & Brand, M.D. (1999) Eur. J. Biochem.263, 671–685], top‐down control analysis was used to describe the control pattern of energy metabolism in rat hepatocytes. The system was divided into nine reaction blocks (glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption) linked by five intermediates (intracellular glucose 6‐phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential). The kinetic responses (elasticities) of reaction blocks to intermediates were determined and used to calculate control coefficients. In the present paper, these elasticities and control coefficients are used to quantify the internal regulatory pathways within the cell. Flux control coefficients were partitioned to give partial flux control coefficients. These describe how strongly one block of reactions controls the flux through another via its effects on the concentration of a particular intermediate. Most flux control coefficients were the sum of positive and negative partial effects acting through different intermediates; these partial effects could be large compared to the final control strength. An important result was the breakdown of the way ATP consumption controlled respiration: changes in ATP level were more important than changes in mitochondrial membrane potential in stimulating oxygen consumption when ATP consumption increased. The partial internal response coefficients to changes in each intermediate were also calculated; they describe how steady state concentrations of intermediates are maintained. Increases in mitochondrial membrane potential were opposed mostly by decreased supply, whereas increases in glucose‐6‐phosphate, NADH/NAD and pyruvate were opposed mostly by increased consumption. Increases in ATP were opposed significantly by both decreased supply and increased consumption.
Biochemical and molecular characterization of the [NiFe] hydrogenase from the hyperthermophilic archaeon, Thermococcus litoralis
Authors: Rákhely Gábor , Zhou Zhi H. , Adams Michael W. W. , Kovács Kornél L.
Published: March 2003

Thermococcus litoralis is a hyperthermophilic archaeon that grows at temperatures up to 98 °C by fermentative metabolism and reduces elemental sulfur (S0) to H2S. A [NiFe] hydrogenase, responsible for H2S or H2 production, has been purified and characterized. The enzyme is composed of four subunits with molecular mass 46, 42, 34 and 32 kDa. Elemental analyses gave approximate values of 22 Fe, 22 S and 1 Ni per hydrogenase. EPR spectra at 70 and 5 K indicated the presence of four or five [4Fe‐4S] and one [2Fe‐2S] type clusters. The optimal temperature for both H2 evolution and oxidation, using artificial electron carriers, was around 80 °C. The operon encoding the T. litoralis enzyme is composed of four genes forming one transcriptional unit, and transcription is not regulated by S0. An unusual transcription‐initiation site is located 139 bp upstream from the translational start point. Sequence analyses indicated the presence of new putative nucleotide‐binding domains. Upstream from the hydrogenase operon, ORFs probably encoding a molybdopterin oxidoreductase enzyme have been identified. Based on sequence, biochemical and biophysical analyses, a model of the enzyme and the pathway of electron flow during catalysis is proposed.
Role of the Src homology 2 domains and interdomain regions in ZAP‐70 phosphorylation and enzymatic activity
Authors: Magistrelli Giovanni , Bosotti Roberta , Valsasina Barbara , Visco Carlo , Perego Rita , Toma Salvatore , Acuto Oreste , Isacchi Antonella
Published: March 2003

The protein tyrosine kinase ZAP‐70, which mediates T‐cell antigen receptor (TCR) signalling, contains three distinct functional modules, two tandemly arranged SH2 domains, a kinase domain and a linker region (interdomain B) that connects them. ZAP‐70 enzymatic activation is strictly dependent on the binding, via its SH2 domains, to the triggered TCR and on tyrosine phosphorylation. Here we utilized recombinant ZAP‐70 and carried out a mutational analysis to understand the structural requirements for its activation. We show that deletion of both SH2 domains corresponding to the first 254 residues moderately increases ZAP‐70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP‐70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320–619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP‐70 to the TCR promotes, through conformational changes, its extensive phosphorylation on tyrosine. However, Tyr292, 315 and 319 do not affect ZAP‐70 enzymatic activity and may influence ZAP‐70 signalling only indirectly by mediating its association with intracellular transducers.
α(1,2)‐Fucosylation prevents sialyl Lewis x expression and E‐selectin‐mediated adhesion of fucosyltransferase VII‐transfected cells
Authors: Zerfaoui Mourad , Fukuda Minoru , Sbarra Véronique , Lombardo Dominique , El‐Battari Assou
Published: March 2003

E‐selectin is a cytokine‐inducible, calcium‐dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte–endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E‐selectin binds to ligands carrying the tetrasaccharide sialyl‐Lewis x (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAc)1 or its isomer sialyl‐Lewis a (NeuAcα2, 3Galβ1,3[Fucα1,4]GlcNAc). We examined the effect of expressing the H‐type α(1,2)‐fucosyltransferase or the α(2,6)‐sialyltransferase on the synthesis of sialyl‐Lewis x by α(1,3)fucosyltransferase. We found that H‐type α(1,2)‐fucosyltransferase but not α(2,6)‐sialyltransferase, strongly inhibited sialyl‐Lewis x expression and E‐selectin adhesion. We assume that H‐type α(1,2)‐fucosyltransferase competes with the endogenous α(2,3)‐sialyltransferase for the N‐acetyllactosamine structures assigned to further serve as acceptors for α(1,3)fucosyltransferase.
The D‐loop structure of human mtDNA is destabilized directly by 1‐methyl‐4‐phenylpyridinium ion (MPP+), a parkinsonism‐causing toxin
Authors: Umeda Shuyo , Muta Tsuyoshi , Ohsato Takashi , Takamatsu Chihiro , Hamasaki Naotaka , Kang Dongchon
Published: March 2003

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine has been reported to cause parkinsonism via its neurotoxic form, 1‐methyl‐4‐phenylpyridinium ion (MPP+), which inhibits complex I of the mitochondrial respiratory chain. Its parkinsonism‐causing mechanisms attract a great deal of interest as a model of the disease. Recently, we reported that MPP+ strongly decreases the amount of mtDNA independent of the inhibition of complex I. Maintenance of a proper amount of mtDNA is essential for the normal function of mitochondria as exemplified in many mitochondrial diseases. The most characteristic feature in vertebral mtDNA replication is that H‐strand synthesis proceeds displacing the parental H‐strand as a long single strand. It forms the D‐loop, a triplex replication intermediate composed of the parental L‐strand, nascent H‐strand and displaced H‐strand. Here we show that MPP+ does not inhibit DNA synthesis by DNA polymerase γ, but rather releases the nascent H‐strands from mtDNA both in organello and in vitro. This indicates that MPP+ directly destabilizes the D‐loop structure, thereby inhibiting replication. This study raises a new mechanism, i.e. destabilization of replication intermediates, for depletion of mtDNA.