bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of HIV‐2 primer–template initiation complex using antisense oligonucleotides
Authors: Boulmé Florence , Freund Frédéric , Gryaznov Sergei , Nielsen Peter E. , Tarrago‐Litvak Laura , Litvak Simon
Published: March 2003

HIV‐2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5′‐region of viral RNA. The HIV‐2 genome has two A‐rich regions upstream of the primer binding site. In contrast to HIV‐1 RNA, no direct evidence of interactions with the U‐rich anticodon loop of tRNALys3 has been described to date. Here we address the question of the potential role of the interactions between these highly structured regions in the initiation of viral DNA synthesis. To evaluate this we used an antisense approach, first validated in our in vitro HIV‐1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre‐primer binding site (the upstream region contiguous to the HIV‐2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A‐rich loop of viral RNA led to an important destabilization of the pre‐primer binding site; this region became accessible to anti‐pre‐primer binding site oligonucleotides in a cooperative manner. These studies allowed to identify an A‐rich region in HIV‐2ROD RNA capable of interacting with tRNALys3. Better knowledge of these interactions is very important for understanding the primer/template positioning in the early steps of HIV‐2 reverse transcription.
The 28.3 kDa FK506 binding protein from a thermophilic archaeum, Methanobacterium thermoautotrophicum, protects the denaturation of proteins in vitro
Authors: Ideno Akira , Yoshida Takao , Furutani Masahiro , Maruyama Tadashi
Published: March 2003

Two families of FK506 binding protein (FKBP) type peptidyl‐prolyl cis–trans isomerase (PPIase) have been found in Archaea. One is the 16–18 kDa short type FKBP family, and another is the 26–30 kDa long type FKBP family. The latter has a longer C‐terminal region than the former. In this study, the 28.3 kDa long type FKBP gene from a thermophilic archaeum, Methanobacterium thermoautotrophicum, was expressed in Escherichia coli, and its gene product (MbFK) was characterized. The PPIase activity of MbFK was much lower than those of other FKBPs reported against oligopeptidyl substrates. MbFK protected green fluorescent protein (GFP) and rhodanese from thermal denaturation. Furthermore, MbFK suppressed the aggregation of chemically unfolded rhodanese and elevated the yield of its refolding although this activity was weaker than that of GroEL/ES. We made two deletion mutants, MbFK‐N which lacked the C‐terminal region, and MbFK‐C which had only the C‐terminal region. Far‐UV CD spectra of these mutants showed that their secondary structures did not change from that of the wild‐type. Whereas the PPIase activity of MbFK‐N was low but detectable, that of MbFK‐C was undetectable. The MbFK‐C protected the thermal protein aggregation, and possessed a weak but significant aggregation suppressing activity against chemically unfolded protein. However, the MbFK‐N did not suppress the aggregation of chemically unfolded rhodanese while it protected heat induced aggregation of rhodanese. These results may indicate that aggregation suppressing activity of MbFK‐W against chemically unfolded protein are exerted mainly by its C‐terminal domain while both domains contribute to thermal protein aggregation suppression.
Perlecan domain V of Drosophila melanogaster
Authors: Friedrich Martin V. K. , Schneider Martina , Timpl Rupert , Baumgartner Stefan
Published: March 2003

The C‐terminal domain V of the basement membrane proteoglycan perlecan was previously shown to play a major role in extracellular matrix and cell interactions. A homologous sequence of 708 amino‐acid residues from Drosophila has now been shown to be 33% identical to mouse perlecan domain V. It consists of three laminin G‐type (LG) and epidermal growth factor‐like (EG) modules but lacks the EG3 module and a link region found in mammalian perlecans. Recombinant production of Drosophila perlecan domain V in mammalian cells yielded a 100‐kDa protein which was folded into a linear array of three globular LG domains. Unlike the mouse counterpart, domain V from Drosophila was not modified by glycosaminoglycans and endogenous proteolysis, due to the absence of the link region. It showed moderate affinities for heparin and sulfatides but did not bind to chick α‐dystroglycan or to various mammalian basement membrane proteins. A single RGD sequence in LG3 of Drosophila domain V was also incapable of mediating cell adhesion. Production of a proteoglycan form of perlecan (≈ 450 kDa) in one Drosophila cell line could be demonstrated by immunoblotting with antibodies against Drosophila domain V. A strong expression was also found by in situ hybridization and immunohistology at various stages of embryonic development and expression was localized to several basement membrane zones. This indicates, as for mammalian species, a distinct role of perlecan during Drosophila development.
Towards a molecular understanding of polycythemia rubra vera
Authors: Pahl Heike L.
Published: March 2003

Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.
The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251
Authors: van der Veen Bart A. , Uitdehaag Joost C. M. , Dijkstra Bauke W. , Dijkhuizen Lubbert
Published: March 2003

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active‐site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products.

Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins.

Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47→Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47→Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity.
A novel NADPH:diamide oxidoreductase activity in Arabidopsis thaliana P1 ζ‐crystallin
Authors: Mano Jun’ichi , Babiychuk Elena , Belles‐Boix Enric , Hiratake Jun , Kimura Akira , Inzé Dirk , Kushnir Sergei , Asada Kozi
Published: March 2003

The ζ‐crystallin (ZCr) gene P1 of Arabidopsis thaliana, known to confer tolerance toward the oxidizing drug 1,1′‐azobis(N,N‐dimethylformamide) (diamide) to yeast [Babiychuk, E., Kushnir, S., Belles‐Boix, E., Van Montagu, M. & Inzé, D. (1995) J. Biol. Chem. 270, 26224], was expressed in Escherichia coli to characterize biochemical properties of the P1‐ζ‐crystallin (P1‐ZCr). Recombinant P1‐ZCr, a noncovalent dimer, showed NADPH:quinone oxidoreductase activity with specificity to quinones similar to that of guinea‐pig ZCr. P1‐ZCr also catalyzed the divalent reduction of diamide to 1,2‐bis(N,N‐dimethylcarbamoyl)hydrazine, with a kcat comparable with that for quinones. Two other azodicarbonyl compounds also served as substrates of P1‐ZCr. Guinea‐pig ZCr, however, did not catalyze the azodicarbonyl reduction. Hence, plant ZCr is distinct from mammalian ZCr, and can be referred to as NADPH:azodicarbonyl/quinone reductase. The quinone‐reducing reaction was accompanied by radical chain reactions to produce superoxide radicals, while the azodicarbonyl‐reducing reaction was not. Specificity to NADPH, as judged by kcat/Km, was > 1000‐fold higher than that to NADH both for quinones and diamide. N‐Ethylmaleimide and p‐chloromercuribenzoic acid inhibited both quinone‐reducing and diamide‐reducing activities. Both NADPH and NADP+ suppressed the inhibition, but NADH did not, suggesting that sulfhydryl groups reside in the binding site for the phosphate group on the adenosine moiety of NADPH. The diamide‐reducing activity of P1‐ZCr accounts for the tolerance of P1‐overexpressing yeast to diamide. Other possible physiological functions of P1‐ZCr in plants are discussed.
Identification of novel periviscerokinins from single neurohaemal release sites in insects
Authors: Predel Reinhard , Kellner Roland , Baggerman Geert , Steinmetzer Torsten , Schoofs Liliane
Published: March 2003

Three novel members of the periviscerokinin family could be identified directly from extracts of single abdominal perisympathetic organs of blaberoid cockroaches by means of electrospray ionization‐quadrupole time of flight (ESI‐QTOF) MS. Sequences of these periviscerokinins were confirmed by Edman degradation. Their primary structures are GSSGLIPFGRT‐NH2 (Lem‐PVK‐1), GSSGLISMPRV‐NH2 (Lem‐PVK‐2), and GSSGMIPFPRV‐NH2 (Lem‐PVK‐3). Hitherto only known from the American cockroach, this neuropeptide family contains a highly conserved N‐terminus whereas, at the C‐terminus, only the penultimate amino‐acid residue (Arg) has been found in all members of this peptide family. The identified periviscerokinins are the only abundant myoactive peptides in abdominal perisympathetic organs of blaberoid cockroches and they appear to be absent in the retrocerebral complex. Screening of extracts of single abdominal perisympathetic organs (70–90 µm in diameter), from five different species of the suborder Blaberoidea, revealed that they all contain the three neuropeptides which are described here for the first time.
A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase
Authors: Brizio Carmen , Otto Annegret , Brandsch Roderich , Passarella Salvatore , Barile Maria
Published: March 2003

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE‐purified matrix fraction. Apo‐Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE‐purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.
Immunohistochemical evidence for the myeloperoxidase/H2O2/halide system in human atherosclerotic lesions
Authors: Malle Ernst , Waeg Georg , Schreiber Renate , Gröne Elisabeth F. , Sattler Wolfgang , Gröne Hermann‐Josef
Published: March 2003

The ‘oxidation theory’ of atherosclerosis proposes that oxidation of low density lipoprotein (LDL) contributes to atherogenesis. Although the precise mechanisms of in vivo oxidation are widely unknown, increasing evidence suggests that myeloperoxidase (MPO, EC 1.11.1.7), a protein secreted by activated phagocytes, generates modified/oxidized (lipo)proteins via intermediate formation of hypochlorous acid (HOCl). In vitro generation of HOCl transforms lipoproteins into high uptake forms for macrophages giving rise to cholesterol‐engorged foam cells. To identify HOCl‐modified‐epitopes in human plaque tissues we have raised monoclonal antibodies (directed against human HOCl‐modified LDL) that do not cross‐react with other LDL modifications, i.e. peroxynitrite‐LDL, hemin‐LDL, Cu2+‐oxidized LDL, 4‐hydroxynonenal‐LDL, malondialdehyde‐LDL, glycated‐LDL, and acetylated‐LDL. The antibodies recognized a specific epitope present on various proteins after treatment with OCl− added as reagent or generated by the MPO/H2O2/halide system. Immunohistochemical studies revealed pronounced staining for HOCl‐modified‐epitopes in fibroatheroma (type V) and complicated (type VI) lesions, while no staining was observed in aortae of lesion‐prone location (type I). HOCl‐oxidation‐specific epitopes are detected in cells in the majority of atherosclerotic plaques but not in control segments. Staining was shown to be inside and outside monocytes/macrophages, endothelial cells, as well as in the extracellular matrix. A similar staining pattern using immunohistochemistry could be obtained for MPO. The colocalization of immunoreactive MPO and HOCl‐modified‐epitopes in serial sections of human atheroma (type IV), fibroatheroma (type V) and complicated (type VI) lesions provides further convincing evidence for MPO/H2O2/halide system‐mediated oxidation of (lipo)proteins under in vivo conditions. We propose that MPO could act as an important link between the development of atherosclerotic plaque in the artery wall and chronic inflammatory events.
Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus
Authors: Hirooka Kazutake , Ohnuma Shin‐ichi , Koike‐Takeshita Ayumi , Koyama Tanetoshi , Nishino Tokuzo
Published: March 2003

A member of the medium‐chain prenyl diphosphate synthases, Bacillus stearothermophilus heptaprenyl diphosphate synthase, catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce (all‐E)‐C35 prenyl diphosphate as the ultimate product. We previously showed that the product specificity of short‐chain prenyl diphosphate synthases is regulated by the structure around the first aspartate‐rich motif (FARM). The FARM is also conserved in a subunit of heptaprenyl diphosphate synthase, component II′, which suggests that the structure around the FARM of component II′ regulates the elongation. To determine whether component II′ regulates the product chain length by a mode similar to that of the short‐chain prenyl diphosphate synthases, we replaced a bulky amino acid at the eighth position before the FARM of component II′, isoleucine 76, by glycine and analyzed the product specificity. The mutated enzyme, I76G, can catalyze condensations of isopentenyl diphosphate beyond the native chain length of C35. Moreover, two mutated enzymes of A79Y and S80F, which have a single replacement to the aromatic residue at the fourth or the fifth position before the FARM, mainly yielded a C20 product. These results strongly suggest that a common mechanism controls the product chain length of both short‐chain and medium‐chain prenyl diphosphate synthases and that, in wild‐type heptaprenyl diphosphate synthase, the prenyl chain can grow on the surface of the small residues at positions 79 and 80, and the elongation is precisely blocked at the length of C35 by isoleucine 76.
Structure/thermodynamics relationships of lectin–saccharide complexes
Authors: Bradbrook Gail M. , Forshaw Jeffrey R. , Pérez Serge
Published: March 2003

Molecular dynamics (MD) simulations of Erythrina corallodendron lectin binding to a monosaccharide, α‐galactose, and a disaccharide, N‐acetyl lactosamine, have been performed in order to investigate the relationship between structure and thermodynamics.

A simulated annealing protocol has been used to generate ensembles of structures for the two complexes, from which both qualitative and quantitative information on binding dynamics have been extracted. The ensembled averaged lectin–saccharide interaction enthalpy is equivalent for both sugars, whereas the calculation based on the X‐ray structures does show a difference. Within large statistical errors, the calculated ‘binding enthalpy’ is also the same for the two systems. These errors arise largely from terms involving solvent and are a typical limitation of current MD simulations.

Significant qualitative differences in binding between the two complexes are, however, observed over the ensembles. These could be important for unraveling the structure/thermodynamic relationship. Stated simply, there are a greater number of binding options available to the disaccharide compared to the monosaccharide. The implications of alternative binding states on thermodynamic parameters and the ‘breaking of enthalpy–entropy compensation’ are discussed. The role of solvent in lectin–saccharide complex formation is suggested to be significant.
Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin‐A can be produced in human kidney 293 cellsbut not in Chinese hamster ovary cells
Authors: Van den Nieuwenhof Ingrid M. , Koistinen Hannu , Easton Richard L. , Koistinen Riitta , Kämäräinen Meerit , Morris Howard R. , van Die Irma , Seppälä Markku , Dell Anne , Van den Eijnden Dirk H.
Published: March 2003

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO‐type glycans and is devoid of any of the N,N′‐diacetyllactosediamine (lacdiNAc)‐based chains previously identified in glycodelin‐A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a β1→4‐N‐acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc‐based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc‐based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic‐type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.
Valyl‐tRNA synthetase from Escherichia coli
Authors: Hountondji Codjo , Beauvallet Christian , Dessen Philippe , Hoang‐Naudin Chau , Schmitter Jean‐Marie , Pernollet Jean‐Claude , Blanquet Sylvain
Published: March 2003

Bromomethyl ketone derivatives of l‐valine (VBMK), l‐isoleucine (IBMK), l‐norleucine (NleBMK) and l‐phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl‐tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for l‐valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl‐bromomethyl ketone (VBMK) resulted in a complete loss of valine‐dependent isotopic [32P]PPi–ATP exchange activity. l‐Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl‐, norleucyl‐ and phenylalanyl‐bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix‐assisted laser desorption‐ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino‐acid‐derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino‐acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259–291 and 419–434 in the primary structure of E. coli ValRS. In fragment 419–434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific l‐valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259–291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259–291 with all the available ValRS amino‐acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.
Mass spectrometry study of ecto‐5′‐nucleotidase from bull seminal plasma
Authors: Fini Carlo , Amoresano Angela , Andolfo Annapaola , D’Auria Sabato , Floridi Ardesio , Paolini Sara , Pucci Piero
Published: March 2003

The structure of ecto‐5′‐nucleotidase from bull seminal plasma, containing a glycosyl‐phosphatidylinositol anchor, was studied using mass spectrometry. MALDI‐MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for ≈ 6000 Da. MALDI‐MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N‐glycosylation. GC‐MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high‐mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high‐mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC‐MS analysis, detailed characterization of the glycosyl‐phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl‐phosphatidylinositol core contained EtN(P)Man3GlcNH2‐myo‐inositol(P)‐glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1‐palmitoylglycerol and 1‐stearoylglycerol outweighed 2‐palmitoylglycerol and 2‐stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S–S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51–Cys57, Cys353–Cys358, Cys365–Cys387 and Cys476–Cys479. This work resolves details of the structure of ecto‐5′‐nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl‐phosphatidylinositol anchor.
Calcium transport across the sarcoplasmic reticulum
Authors: Stokes David L. , Wagenknecht Terence
Published: March 2003

Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic‐reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+‐ATPase effects reuptake in an ATP‐dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+‐ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+‐ATPase at 8‐Å resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP‐binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30‐Å resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling.
Flavin–protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C‐ and 15N‐labelled flavin
Authors: Fleischmann Garrit , Lederer Florence , Müller Franz , Bacher Adelbert , Rüterjans Heinz
Published: March 2003

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C‐ and 15N‐labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen‐bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the π‐electron distribution.

The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N‐chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C‐NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N‐H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins.

With respect to N(10), it is more sp3‐hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3‐hybridized than in free FMN, but N(5) exhibits a higher degree of sp3‐hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane.

In addition, two‐electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N‐chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins.

31P‐NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.
Interaction of antimony tartrate with the tripeptide glutathione
Authors: Sun Hongzhe , Yan Siu Cheong , Cheng Wing Shan
Published: March 2003

The tripeptide glutathione (γ‐l‐Glu‐l‐Cys‐Gly, GSH) is thought to play an important role in the biological processing of antimony drugs. We have studied the complexation of the antileishmanial drug potassium antimony(III) tartrate to GSH in both aqueous solution and intact red blood cells by NMR spectroscopy and electrospray ionization mass spectrometry. The deprotonated thiol group of the cysteine residue is shown to be the only binding site for Sb(III), and a complex with the stoichiometry [Sb(GS)3] is formed. The stability constant for [Sb(GS)3] was determined to be logK25 (I = 0.1 m, 298 K) based on a competition reaction between tartrate and GSH at different pH* values. In spite of being highly thermodynamically stable, the complex is kinetically labile. The rate of exchange of GSH between its free and Sb‐bound form is pH‐dependent, ranging from slow exchange on the1H‐NMR timescale at low pH (2 s−1at pH 3.2) to relatively rapid exchange at biological pH (> 440 s−1). Such facile exchange may be important in the transport of Sb(III) in various biofluids and tissuesin vivo. Our spin–echo1H‐NMR data show that Sb(III) rapidly entered red blood cell walls and was complexed by intracellular glutathione.
Symmetry and structure in P‐glycoprotein and ABC transporters
Authors: Jones Peter M. , George Anthony M.
Published: March 2003

The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched. ABC proteins are involved in the trafficking of a diverse variety of biological molecules across cell membranes, with some members implicated in medical syndromes such as cystic fibrosis and multidrug resistance to anti‐cancer drugs. In the absence of X‐ray crystallographic data, structural information has come from spectroscopy, electron microscopy, secondary structure prediction algorithms and residue substitution, epitope labelling and cysteine cross‐linking studies. These have generally supported a model for the topology of the transmembrane domains of ABC transporters in which a single aqueous pore is formed by a toroidal ring of 12 α helices, deployed in two arcs of six helices each. Although this so‐called 6 + 6 helix model can be arranged in either mirror or rotational symmetry configurations, experimental data supports the former. In this review, we put forward arguments against both configurations of this 6 + 6 helix model, based on what is known generally about symmetry relationships in proteins. We relate these arguments to P‐glycoprotein, in particular, and discuss alternative models for the structure of ABC transporters in the light of the most recent research.
The helix nucleation site and propensity of the synthetic mitochondrial presequence of ornithine carbamoyltransferase
Authors: de Jongh Harmen H. J.
Published: March 2003

This study describes the helix nucleation site and helix propagation of the amphiphilic helical structure of the mitochondrial presequence of rat ornithine carbamoyltransferase. We investigated this property of the 32‐residue synthetic presequence using CD and 2D‐HR NMR techniques by determining the structure as a function of the concentration of trifluoroethanol. It was found that the hydrophobic cluster Ile7‐Leu8‐Leu9 forms the helix nucleation site, expanding to include residues Asn4 to Lys16 when the concentration of trifluoroethanol is increased from 10 to 30%. At higher trifluoroethanol concentrations an increased ‘stiffening’ of the polypeptide backbone (to Arg26) is observed. In addition, by recording CD spectra at different trifluoroethanol concentrations as a function of temperature, it was found that the equilibrium constant between helix and random coil formation for this peptide exhibits a strong temperature dependence with maximum values between 20 and 30 °C. Comparison of these equilibrium constants with those of homopolymers stressed the unique character of the mitochondrial presequence. The findings are discussed in relation to the molecular recognition events at different stages of the transport process of this protein into mitochondria.
Apoptosis induced by oxidized low density lipoprotein in human monocyte‐derived macrophages involves CD36 and activation of caspase‐3
Authors: Wintergerst Eva S. , Jelk Jennifer , Rahner Christoph , Asmis Reto
Published: March 2003

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)‐induced apoptosis in human monocyte‐derived macrophages. Anti‐CD36 mAb SMO and OKM‐5 reduced the number of apoptotic cells in OxLDL‐treated macrophages by more than 94%, but they did not block ceramide‐triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose‐dependent manner with an IC50 of 10–30 µm. OxLDL did not induce apoptosis in CD36‐negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL‐triggered programmed cell death. Neither anti‐CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL‐induced apoptosis did not block the uptake of 3H‐labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H‐labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL‐induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase‐3 activity in human macrophages. Activation of caspase‐3 was blocked by anti‐CD36 Ig and the caspase‐3 inhibitor Z‐DEVD‐FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL‐specific signaling event, which leads to the rapid activation of caspase‐3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.
Interaction of peroxisomes with microtubules
Authors: Thiemann Meinolf , Schrader Michael , Völkl Alfred , Baumgart Eveline , Fahimi H. Dariush
Published: March 2003

The association of membrane‐bounded cell organelles to microtubules is crucial for determination of their shape, intracellular localization and translocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature asin vivostudies indicate that only a few peroxisomes move along the microtubular tracks. In order to characterize the interactions of peroxisomes with microtubules, we have developed a semiquantitativein vitrobinding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules coated onto microtiterplates. The binding was visualized by differential interference contrast and immunofluorescence using a confocal laser scanning microscope. The binding was concentration dependent and saturable, being affected by time, temperature, and pH. Addition of ATP or the motor proteins kinesin and dynein increased the binding capacity, while ATP‐depletion or microtubule associated proteins (MAPs) decreased it. KCl treatment of peroxisomes reduced the binding, which was restored by dialyzed KCl‐stripping eluate as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases orN‐ethylmaleimide. Moreover, the treatment of peroxisomes with proteases orN‐ethylmaleimide reduced their binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in the binding of peroxisomes to microtubules. This notion is supported by the observation that distinct subfractions of dialyzed KCl‐stripping eluate obtained by gel chromatography augmented the binding. Those subfractions, as well as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)‐115, revealing a 70‐kDa polypeptide. Moreover, immunodepletion of KCl‐stripping eluate and its subfractions with an antibody to the conserved microtubule binding domain of CLIPs, abolished their promoting effect on the binding, thus suggesting the involvement of a CLIP‐related protein in the binding of peroxisomes to microtubules.
Sterol‐induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double‐bond position in the sterol tetracyclic ring structure
Authors: Leppimäki Petra , Mattinen Jorma , Slotte J. Peter
Published: March 2003

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7‐dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 µm cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol·mg−1 protein (from 90 nmol·mg−1 to 190 nmol·mg−1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol‐enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7‐dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane‐bound CTP:phosphocholine cytidylyltransferase, the rate‐limiting enzyme, was increased by 47 ± 4% in cholesterol‐enriched cells whereas its activity was unchanged in lathosterol‐enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down‐regulated acetate‐incorporation into cholesterol, and upregulated sterol esterification in the sterol‐enriched fibroblasts. Using 31P‐NMR to measure the lamellar‐to‐hexagonal (Lα–HII) phase transition in multilamellar lipid dispersions, lathosterol‐containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine and either cholesterol or lathosterol (70 : 30 mol/mol), differential scanning calorimetry also revealed that the Lα–HII‐transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lα–HII‐transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.
Mutagenesis at Asp27 of pyruvate decarboxylase from Zymomonas mobilis
Authors: Wu Yong‐Ge , Chang Alan K. , Nixon Peter F. , Li Wei , Duggleby Ronald G.
Published: March 2003

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three‐dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by‐product from the hydroxyethyl–ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by‐product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild‐type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30‐fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild‐type. The time‐courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild‐type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild‐type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild‐type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.
Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli
Authors: Incharoensakdi Aran , Matsuda Nobuyuki , Hibino Takashi , Meng Yu‐Ling , Ishikawa Hiroshi , Hara Akira , Funaguma Tohru , Takabe Tetsuko , Takabe Teruhiro
Published: March 2003

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of ω‐aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for ω‐aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for ω‐aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.
The structure of the nonreducing terminal groups in the O‐specific polysaccharides from two strains of Bordetella bronchiseptica
Authors: Vinogradov Evgeny , Peppler Mark S. , Perry Malcolm B.
Published: March 2003

The structures of the polysaccharide chains of the LPS from Bordetella bronchiseptica strains 110H and Bp512 were analysed by NMR spectroscopy and mass spectrometry. The polysaccharides consist of α‐(1–4)‐linked 2,3‐diacetamido‐2,3‐dideoxy‐l‐galacturonic acid repeating units. Polysaccharides from both strains have 2,3,4‐triamino‐2,3,4‐trideoxy‐α‐galacturonamide derivatives at their nonreducing ends, a monosaccharide identified for the first time in nature. The polymers from the two strains differ in the nature of the acylation of the amino groups of this monosaccharide. In the strain 110H, the residue is formylated at positions 3 and 4, and has N‐formyl‐l‐alanyl or l‐alanyl substituents at N‐2. In the strain Bp512, the amino group at position 2 is acetylated, at position 3 it is formylated, and the amino group at position 4 bears a 2‐methoxypropionyl substituent. The distribution of the acyl groups was determined from long range 1H‐13C correlation (HMBC) NMR spectra. Measurement of the spectra under different pH conditions showed that carboxyl groups of the inner uronic acid residues of the polymeric chain are free, and that carboxyl groups of the terminal residues are amidated. These conclusions were confirmed by the results of mass spectrometric analysis.
CB1 receptor–G protein association
Authors: Mukhopadhyay Somnath , Howlett Allyn C.
Published: March 2003

The CB1 cannabinoid receptor in N18TG2 neuroblastoma cells inhibits adenylate cyclase, and this response can be mimicked by a peptide corresponding to the juxtamembrane C‐terminal domain (CB1401–417). Guanosine 5′‐O‐(3‐thio)triphosphate binding to G proteins can be stimulated by both peptide CB1401–417 and peptides corresponding to the third intracellular loop [Howlett, A.C., Song, C., Berglund, B.A., Wilken, G.H. & Pigg, J.J. (1998) Mol. Pharmacol.53, 504–510; Mukhopadhyay, S., Cowsik, S.M., Welsh, W.J. & Howlett, A.C. (1999) Biochemistry38, 3447–3455]. In Chaps‐solubilized N18TG2 membranes, the CB1 receptor coimmunoprecipitated with all three Gi subtypes. Pertussis toxin significantly reduced the CB1 receptor–Gαi association and attenuated the CB1401–417‐induced inhibition of adenylate cyclase. CB1401–417 significantly reduced the CB1 receptor association with Gαi3, but not with Gαi1 or Gαi2. In contrast, third intracellular loop peptides significantly reduced the CB1 receptor association with Gαi1 and Gαi2, but not Gαi3. These interactions are specific for the CB1 receptor because a peptide corresponding to the juxtamembrane C‐terminal domain of the CB2 receptor failed to compete for the association of the CB1 receptor with any of the Giα subtypes, and was not able to activate Gi proteins to inhibit adenylate cyclase. These studies indicate that different domains of the CB1 receptor direct the interaction with specific G protein subtypes.
Major sites for the differentiation of Vα14+ NKT cells inferred from the V‐J junctional sequences of the invariant T‐cell receptor α chain
Authors: Shimamura Michio , Miura‐Ohnuma Jun , Huang Yi‐Ying
Published: March 2003

CD1d‐restricted mouse NK1.1+ TCRαβ+ natural killer T (NKT) cells predominantly use an invariant TCR α chain encoded by Vα14 and Jα281 gene segments with a one‐nucleotide N region. We found that NKT cells generated in the culture of fetal liver precursors possessed Vα14‐Jα281 junctions that could be produced without the action of terminal deoxyribonucleotidyl transferase (TdT), indicating that NKT cells derived from fetal liver precursors are distinguishable from those from adult precursors with TdT expression. In fact, the frequency of the fetal‐form sequences decreased with ageing. Surprisingly, the fetal‐type sequences were predominantly observed in the lymphoid organs of athymic mice with the exception of bone marrow, where a sequence peculiar to the organ, with TdT‐involved conversion from the invariant junction, was frequently present. These findings suggest that there are two independent sites of Vα14+ NKT cell development, the hematopoietic organs throughout life (the developing liver and adult bone marrow) and, principally, the mature thymus.