bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural analysis of oligosaccharide‐alditols released by reductive β‐elimination from the jelly coats of the anuran Bufo arenarum
Authors: Morelle Willy , Cabada Marcelo O. , Strecker Gérard
Published: March 2003

Amphibian egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they are transported toward the cloaca. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O‐linked carbohydrate chains of the jelly coat surrounding the eggs of Bufo arenarum were released by alkaline borohydride treatment. Fractionation of the mixture of O‐linked oligosaccharide‐alditols was achieved by a combination of chromatographic techniques comprising gel‐permeation chromatography, ion‐exchange chromatography and high‐performance liquid chromatography using an amino‐bonded silica column, afforded 11 fractions. The primary structures of these O‐glycans were determined by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy in conjunction with matrix‐assisted laser‐desorption‐ionization−time‐of‐flight mass spectrometry. 11 oligosaccharide structures, possessing a core consisting of Galβ1←3GalNAc‐ol with or without branching through a GlcNAc residue linked β1←6 to the GalNAc residue (core type 2 or core type 1, respectively) are described. These oligosaccharide‐alditols with these types of cores have been identified previously in mammalian mucins or in oviducal amphibian jellies. These glycans contain blood group determinants such as H, A or Cad antigens.
Effect of the extramitochondrial adenine nucleotide pool size on oxidative phosphorylation in isolated rat liver mitochondria
Authors: Schild Lorenz , Gellerich Frank‐Norbert
Published: March 2003

The effect of the concentration of extramitochondrial adenine nucleotides on oxidative phosphorylation was studied in isolated rat liver mitochondria. Mitochondria were incubated with succinate and hexokinase or creatine kinase at constant or varying extramitochondrial adenine nucleotide concentrations ranging over 0.3−5 mM. As parameters of oxidative phosphorylation, rate of respiration, membrane potential as well as intra‐ and extra‐mitochondrial adenine nucleotide concentrations were determined. Below a threshold concentration of extramitochondrial adenine nucleotides of 2 mM, the free Gibb's energy for the adenine nucleotide transport increased but the extramitochondrial ATP/ADP ratio decreased at intermediate rates of respiration with decreasing extramitochondrial adenine nucleotide concentrations. In this range the rate of respiration was dependent on the extramitochondrial ADP concentration. No effect of the extramitochondrial adenine nucleotide concentration on the relationships between the rate of respiration and the membrane potential, the intramitochondrial adenine nucleotide pool and the intramitochondrial ATP/ADP ratio was found. This suggests that the decline of extramitochondrial ATP due to adenine nucleotide degradation and the limitation of adenine nucleotide transport may be of importance in the postischemic phase as nucleotide resynthesis and reorganization of physiological ion distribution are ATP consuming processes
Two vascular apoptosis‐inducing proteins from snake venom are members of the metalloprotease/disintegrin family
Authors: Masuda Shinako , Hayashi Hiroshi , Araki Satohiko
Published: March 2003

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells. In a previous report, we described the purification of a vascular apoptosis‐inducing protein (VAP) from Crotalus atrox [Masuda, S., Araki, S., Kaji, K. & Hayashi, H. (1997) Biochem. Biophys. Res. Commun. 235, 59−63]. We report here the identification of a second vascular apoptosis‐inducing protein, VAP2, in venom from C. atrox. When we fractionated crude venom from C. atrox by isoelectric focusing, we found two proteins with apoptosis‐inducing activity, one was basic and the other acidic. The basic protein corresponded to VAP, and we named the acidic protein VAP2. VAP2 was a monomeric protein with molecular mass of 55 kDa and an isoelectric point of 4.5. VAP2 killed vascular endothelial cells in culture, and the death of cells exhibited the characteristic features of apoptotic activity of VAP2 seemed to be specific to endothelial cells, as reported for VAP. The half‐lethal doses of VAP and VAP2 were 0.3 μg/ml and 0.1 μg/ml, respectively. Analysis of the partial amino acid sequences of VAP and VAP2 revealed similarities to members of the metalloprotease/disintegrin family. The sensitivity of VAP2 to changes in pH and temperature was distinct from that of VAP. Our results suggest that VAP and VAP2 are members of the metalloprotease/disintegrin family.
Cellobiose dehydrogenase enhances Phanerochaete chrysosporium cellobiohydrolase I activity by relieving product inhibition
Authors: Igarashi Kiyohiko , Samejima Masahiro , Eriksson Karl‐Erik L.
Published: March 2003

The interaction of cellobiose dehydrogenase (CDH) with cellobiohydrolase I (CBH I) in cellulose‐grown cultures of Phanerochaete chrysosporium was investigated to clarify the role of CDH in cellulose degradation. Decomposition of bacterial microcrystalline cellulose by CBH I was enhanced significantly in the presence of the CDH/ferricyanide redox‐system compared with CBH I alone. To explain this phenomenon, a model system, using p‐nitrophenyl‐β‐
D‐cellobioside as a substrate, was elaborated for measurement of CBH I activity with and without the CDH redox‐system. The activity of CBH I for hydrolysis of p‐nitrophenyl‐β‐
D‐cellobioside was also enhanced in the presence of the redox system. It was found that Km for hydrolysis of p‐nitrophenyl‐β‐
D‐cellobioside by CBH I was lower in the presence than in the absence of the CDH/ferricyanide redox‐system, 142 μM and 384 μM, respectively, while no significant difference was observed between the kcat values. These results indicate that cellulase activity is enhanced by an increased affinity for p‐nitrophenyl‐β‐
D‐cellobioside, rather than by an increased hydrolysis rate. This shows that cellobiose, the hydrolysis product, acts as a competitive inhibitor of the interaction between CBH I and p‐nitrophenyl‐β‐
D‐cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p‐nitrophenyl‐β‐
D‐cellobioside by CBH I in the absence of the CDH redox system, and the Ki value for cellobiose inhibition was estimated to be 65 μM. However, this inhibition did not occur if cellobiose was incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone.
Oligosaccharides derived by endo‐β‐galactosidase digestion of bovine corneal keratan sulphate
Authors: Huckerby Thomas N. , Tai Gui‐Hua , Nieduszynski Ian A.
Published: March 2003

Bovine corneal keratan sulphate has been fragmented using the enzyme endo‐β‐galactosidase and 1H‐NMR chemical shift data are reported for five newly isolated tetrasaccharides which derived from the repeat region. They have the structures : GlcNAc(β1‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal‐ol, GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc(β1‐3)Gal‐ol, GlcNAc(β1‐3)Gal6Sβ(1‐4)GlcNAc6S(β1‐3)Gal‐ol, GlcNAc6S(β1‐3)Gal6S(β1‐4)GlcNAc(β1‐3)Gal‐ol, and GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S(β1‐3)Gal‐ol. Structures for two trisaccharides formed by a peeling reaction are also given. These are GlcNAc(β1‐3)Gal6S(β1‐4)GlcNAc‐ol and GlcNAc6S(β1‐3)Gal(β1‐4)GlcNAc6S‐ol where GlcNAc6S‐ol represents N‐acetylglucosaminitol 6‐O‐sulphate.

Characterisation of such structures and their spectral assignments will be of considerable value for the studies of both selectin ligands and undersulphated keratan sulphates such as those occurring on cell surfaces and in brain tissue.
Structure and regulation of the bifunctional enzyme lysine‐oxoglutarate reductase‐saccharopine dehydrogenase in maize
Authors: Kemper Edson L. , Cord‐Neto Germano , Capella Adriana N. , Gonçalves‐Butruile Marymar , Azevedo Ricardo A. , Arruda Paulo
Published: March 2003

The lysine‐oxoglutarate reductase (LOR) domain of the bifunctional enzyme lysine‐oxoglutarate reductase‐saccharopine dehydrogenase (LOR/SDH) from maize endosperm was shown to be activated by Ca2+, high salt concentration, organic solvents and Mg2+. The Ca2+‐dependent enhancement of LOR activity was inhibited by the calmodulin antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W7) and calmidazolium. Limited proteolysis was used to assess the structure/function relationship of the enzyme. Digestion with elastase separated the bifunctional 125‐kDa polypeptide into two polypeptides of 65 kDa and 57 kDa, containing the functional domains of LOR and SDH, respectively. Proteolysis did not affect SDH activity, while LOR showed a time‐dependent and protease‐concentration‐dependent inactivation followed by reactivation. Prolonged digestion or increasing amounts of elastase produced a complex pattern of limit polypeptides derived from additional cleavage sites within the 65‐kDa (LOR) and 57‐kDa (SDH) domains. The SDH‐containing polypeptides inhibited the enzymatic activity of LOR‐containing polypeptides. When separated from the SDH domain by limited proteolysis and ion‐exchange chromatography, the LOR domain retained its Ca2+ activation property, but was no longer activated by high salt concentrations. These results suggest that the LOR activity of the native enzyme is normally inhibited such that after modulation, the enzyme undergoes a conformational alteration to expose the catalytic domain for substrate binding.
The Ulip family phosphoproteins
Authors: Byk Tamara , Ozon Sylvie , Sobel André
Published: March 2003

The search for intracellular phosphoproteins implicated in the regulation of neuronal differentiation led to the identification of Ulip1, a mammalian protein related to the Caenorhabditis elegans unc‐33 gene product [Byk, T., Dobransky, T., Cifuentes‐Diaz, C. & Sobel, A. (1996) J. Neurosc. 16, 688−701]. The expression level and phosphorylation pattern of Ulip1 were shown to be strongly regulated during development and neuronal differentiation. We have isolated three additional complete coding sequences for members of the Ulip family in the mouse, Ulips 2−4, all preferentially expressed in the nervous system. Furthermore, two Ulip sequences, Ulips A and Ulips B, could be identified in C. elegans. The Ulip family is highly conserved throughout evolution (more than 96 % for Ulips 1−3 and 92.5 % for Ulip4 between mouse and human) and the various members of the family within a single species display about 75 % similarity. Sequence comparisons further reveal several highly similar domains and subdomains, including a 32‐amino‐acid region highly conserved from a bacterial hydantoinase to human Ulips. Two‐dimensional immunoblot analysis of in vitro translated Ulips 1−4 demonstrates the existence, for each Ulip protein, of several, most probably differentially phosphorylated forms, in agreement with the presence of conserved phosphorylation consensus sites within their sequences. The expression of Ulips 1−4 mRNAs is differentially regulated during development and nerve‐growth‐factor‐induced neuronal differentiation of PC12 cells. Our results indicate a differential, possibly complementary role of phosphoproteins of the highly conserved Ulip family in the control of neuronal differentiation, in relation with the development and plasticity of the nervous system.
A depressant insect‐selective toxin analog from the venom of the scorpion Leiurus quinquestriatus hebraeus
Authors: Moskowitz Haim , Herrmann Rafael , Jones A. Daniel , Hammock Bruce D.
Published: March 2003

The scorpion venom‐derived excitatory and depressant insect‐selective polypeptide neurotoxins modify sodium conductance in insect neuronal membranes and differ greatly in their primary structures and symptoms induced in blow fly larvae. We report here the purification and characterization of a new insect selective toxin, LqhIT5. LqhIT5 is more similar to the excitatory toxins in its mode of action and the depressant toxins in its primary structure. This toxin is a single polypeptide composed of 61 amino acids that are cross linked by four disulfide bonds. When LqhIT5 is injected into blow fly larvae, a fast contraction paralysis occurs without depressant activity. No mammalian toxicity was detected by subcutaneous or intracranial injections of this toxin into mice. Sequence comparison of LqhIT5 and known depressant toxins shows a high degree of similarity among the amino acids located on the C‐terminus of the toxins. However, there are some clear differences in the amino acids located close to the N‐terminus of the toxins. By the aid of homology modeling, we demonstrated that these amino acids have the same orientation in the tertiary structure of the molecule and are exposed to the environment. The change in the mode of action of LqhIT5 (no depressant activity) by substitutions of a few amino acids located on a specific exposed area of the toxin shed a new light on the structure/function relationship of scorpion toxins. These results caution that similarity in the mechanism of action of scorpion toxins does not always follow from an overall similarity in sequence.
Transcriptional regulation of the rat glutamine synthetase gene by tumor necrosis factor‐α
Authors: Chakrabarti Ratna
Published: March 2003

The expression of glutamine synthetase (GS) is induced in rat skeletal muscle cells (L‐6) in response to treatment with the inflammatory cytokine tumor necrosis factor α (TNF‐α). This paper reports the regulation of GS expression in rat skeletal muscle which expresses high levels of GS. TNF‐α treatment leads to a 3−4‐fold increase in GS activity in a dose‐dependent and time‐dependent manner. Northern‐blot analysis of GS mRNA revealed an increased mRNA concentration, reaching a peak at 12 h in response to TNF‐α treatment. To monitor transcriptional activation of GS by TNF‐α, and to identify a TNF‐α‐responsive element in the GS promoter, L6 cells were treated with TNF‐α following transfection of GS−chloramphenicol‐acetyltransferase (CAT) constructs. The first 251‐bp fragment at the GS upstream sequence showed basal promoter activity, but failed to show any TNF‐α‐inducible activity. However, a 2.5−3‐fold induction was noted in constructs extending up to 1.1 kb. This data demonstrates that the rat GS gene is transcriptionally regulated by TNF‐α and identifies a TNF‐α‐responsive region at the 5′ flanking sequence of the GS gene.
Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276
Authors: Gallagher Stephen C. , George Ashley , Dalton Howard
Published: March 2003

Whole cells of Nocardia corallina B‐276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH‐dependent reductase (reductase) and a regulatory component (coupling protein).

The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence‐alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu‐Xaa‐Xaa‐His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active‐site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.
Pleiotropic phenotype of acetyl‐CoA‐carboxylase‐defective yeast cells
Authors: Hoja Ursula , Wellein Christian , Greiner Eva , Schweizer Eckhart
Published: March 2003

The Saccharomyces cerevisiae gene BPL1 encodes the enzyme biotin :protein ligase (BPL), which is required for acetyl‐CoA carboxylase (ACC) holoenzyme formation. Disruption of one of the two BPL1 alleles present in diploid cells results, upon sporulation, in a 2+ : 20 segregation of cell viability, with none of the two viable spores being BPL1 negative. In contrast to BPL1 deletants, BPL1 base‐substitution mutants are potentially viable and may be isolated as long‐chain‐fatty‐acid‐requiring auxotrophs. In addition to ACC pyruvate carboxylase and an additional biotin‐containing protein of unknown function fail to be biotinylated in BPL1‐defective yeast mutants. In this study, one of these mutants, bpl1‐C25/17, is shown to contain an amber stop codon at position 151 of the 689‐amino‐acid BPL sequence. In bpl1‐C25/17 cells, de novo fatty acid synthesis is almost absent (< 2 % of the wild type), while very‐long‐chain fatty acid (VLCFA) synthesis and, to some extent, medium−long‐chain fatty acid elongation are still active. Hence, endogenous malonyl‐CoA synthesis is reduced but not abolished by the translational stop mutation. A low rate of intact‐BPL synthesis is accomplished in the mutant by occasional readthrough of the bpl1‐C25/17 UAG nonsense triplet by normal yeast tRNA.esGln.rb.eiCAG.rb. Correspondingly, ACC biotinylation is severely reduced though not completely absent in the two bpl1 mutants studied in this work. Residual BPL1 expression in bpl1‐C25/17 cells is increased to a level allowing wild‐type‐like growth by transformation with high copy numbers of either the wild‐type tRNA.esGln.rb.eiCAG.rb or the mutant bpl1‐C25/17 genes. It is concluded that the lethality of BPL1 deletants is due to the lack of malonyl‐CoA−dependent VLCFA synthesis and that the viability of distinct ACC‐defective point mutants is due to their maintenance of a critical level of malonyl‐CoA and, hence, VLCFA production. The residual capacity of malonyl‐CoA synthesis, though, is inadequate to allow cytoplasmic bulk de novo fatty acid synthesis, nor does it support mutant growth on 13 : 0 as the only dietary fatty acid. ACC‐defective mutants are respiratory deficient, which is attributed to the failure of mitochondrial fatty acid synthesis. Since lipoic acid levels of ACC1 and BPL1 mutants are essentially normal, an unknown product of mitochondrial fatty acid synthesis appears to be critically reduced in malonyl‐CoA−deficient yeast cells.
The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray‐ionisation mass spectrometry
Authors: Buzy Armelle , Millar Alan L. , Legros Veronique , Wilkins Patricia C. , Dalton Howard , Jennings Keith R.
Published: March 2003

The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray‐ionisation mass spectrometry (ESI‐MS) and liquid chromatography ESI‐MS (LC/ESI‐MS). The hydroxylase is a non‐heme diiron protein consisting of three pairs of non‐identical subunits (α ≈ 60 kDa, β ≈ 45 kDa and γ ≈ 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the α‐subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI‐MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI‐MS results also showed that the α‐subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI‐MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins.
Insights into the molecular basis of thermal stability from the analysis of ion‐pair networks in the Glutamate Dehydrogenase family
Authors: Yip Kitty S. P. , Britton K. Linda , Stillman Timothy J. , Lebbink Joyce , de Vos Willem M. , Robb Frank T. , Vetriani Constantino , Maeder Dennis , Rice David W.
Published: March 2003

The recent structure determination of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus and the comparison of this structure with its counterparts from the mesophiles Clostridium symbiosum and Escherichia coli has highlighted the formation of extended networks of ion‐pairs as a possible explanation for the superior thermal stability of the hyperthermostable enzyme. In the light of this, we have carried out a homology‐based modelling study using sequences of a range of glutamate dehydrogenases drawn from species which span a wide spectrum of optimal growth temperatures. We have attempted to analyse the extent of the formation of ion‐pair networks in these different enzymes and tried to correlate this with the observed thermal stability. The results of this analysis indicate that the ion‐pair networks become more fragmented as the temperature stability of the enzyme decreases and are consistent with a role for the involvement of such networks in the adaptation of enzymes to extreme temperatures.
Sechiumin, a ribosome‐inactivating protein from the edible gourd, Sechium edule Swartz
Authors: Wu Tsann‐Huei , Chow Lu‐Ping , Lin Jung‐Yaw
Published: March 2003

A new ribosome‐inactivating protein (RIP), sechiumin, was purified from the seeds of edible gourd, Sechium edule Swartz by gel‐filtration and ion‐exchange chromatography, with an apparent relative molecular mass of 27 kDa. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50 % inhibition (IC50) of 0.7 nM, but has a much lower inhibitory effect on intact HeLa cells, with an IC50 of 5000 nM. Sechiumin has a highly specific RNA N‐glycosidase activity towards 28S rRNA, as does the A‐chain of abrin. It suggests that sechiumin is one of the type‐I ribosome‐inactivating proteins. The cDNA of sechiumin has been cloned and expressed using a pET expression system in Escherichia coli. The sechiumin cDNA has 951 nucleotides, encoding a protein with 285 amino acids. The amino acid sequence deduced from the cDNA reveals that the first 21 N‐terminal amino acid residues constitutes a signal peptide. Sechiumin has nearly 60 % similarity to several type‐I RIPs purified from the Cucurbitaceae family, such as luffin‐a (62.5 %) and trichosanthin (64.8 %), but less similarity to other type‐I RIPs. Two amino acid residues, Glu160 and Arg163, at the putative active site of sechiumin, are known to be catalytically active in ricin and abrin. The N‐terminal amino acid sequence of sechiumin is very similar to that of trichosanthin. The recombinant sechiumin was obtained as an insoluble protein, and the preparation of the active soluble form was achieved by renaturing the denatured protein. These studies suggest that the recombinant sechiumin could be used for the preparation of immunotoxin as a potential cancer chemotherapeutic agent.
Biochemical evidence for a calmodulin‐stimulated calcium‐dependent protein kinase in maize
Authors: Pandey Sona , Sopory Sudhir K.
Published: March 2003

We provide biochemical evidence for the presence of a Ca2+‐dependent calmodulin (CaM)‐stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE‐Sephacel chromatography, CaM‐Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg2+ for activity and had an optimum pH of 7.5. The kinase is a Ca2+‐binding protein, as was evident by 45Ca2+‐binding and Ca2+ mobility‐gel‐shift assays. 1 μM Ca2+ stimulated the kinase activity about 12‐fold and was further stimulated by the addition of exogenous CaM (≈100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide‐2, histone IIIS and casein. Syntide‐2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca2+ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross‐reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide‐2 and histone IIIS)‐phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.
Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei
Authors: Wille Ulrike , Schade Babette , Duszenko Michael
Published: March 2003

Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms : firstly by a facilitated‐diffusion carrier showing a Km of 0.17 mM and a Vmax of 44 nmol 10−8 cells min−1 that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton‐motive‐force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74 % decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T. brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.
Monofunctional platinum amine complexes destabilize DNA significantly
Authors: Bauer Cornelia , Peleg‐Shulman Tal , Gibson Dan , Wang Andrew H.‐J.
Published: March 2003

Both cis‐[Pt(NH3)2(4‐Me‐Py)Cl]+ and trans‐[Pt(NH3)2(4‐Me‐Py)Cl]+ bind DNA covalently at the N7 site of guanine residues forming mono‐dentate adducts. However, like cisplatin and transplatin, only the cis isomer has anti‐cancer activity, whereas the trans‐isomer does not. In order to understand the molecular basis of the different activities associated with cis‐[Pt(NH3)2(4‐Me‐Py)Cl]+ and trans‐[Pt(NH3)2(4‐Me‐Py)Cl]+, the interactions of these two platinum compounds with the DNA heptamer CCTG*TCC :GGACAGG duplex (G* is the platinated guanine) have been examined. The reaction rate of cis‐[Pt(NH3)2(4‐Me‐Py)Cl]+ with the single‐stranded CCTGTCC is significantly faster than that of the trans isomer. The solution structure of the platinum−DNA adducts has been studied by two‐dimensional NMR spectroscopy. Both the cis‐platinum adducts and the trans‐platinum adducts destabilize the DNA duplex significantly. The melting temperature (Tm) of the platinated heptamer duplex is estimated to be 10 +C lower than for the unplatinated duplex by NMR. At 2 +C, the base pairs located on the 5′ side of the oligonucleotide, beyond the platinum lesion site, are disrupted. Over time, the platinum−DNA complex decomposes and the cis‐[Pt(NH3)2(4‐Me‐Py)] platinum complex is gradually detached from DNA. No interstrand crosslinking is observed. The biological implications of the structural studies are discussed.
Structural characteristics of bullfrog (Rana catesbeiana) transthyretin and its cDNA
Authors: Yamauchi Kiyoshi , Takeuchi Hiro‐aki , Overall Maree , Dziadek Marie , Munro Sharon L. A. , Schreiber Gerhard
Published: March 2003

Transthyretin, an extracellular thyroid‐hormone‐binding protein (THBP) in higher vertebrates, is synthesized and secreted by the choroid plexus of all classes of vertebrates, except fish and amphibians, and synthesized in the liver of endothermic animals. Here, we report the nucleotide sequence of the cDNA for a THBP found in plasma of bullfrog (Rana catesbeiana) tadpoles before the climax of metamorphosis. The amino acid sequence clearly shows this protein to be an amphibian transthyretin. The three‐dimensional structure of bullfrog transthyretin was derived using homology modeling. Compared with transthyretins from other vertebrate species, bullfrog transthyretin is highly conserved at the thyroid hormone‐binding sites and other important structural regions of the subunits. Bullfrog transthyretin mRNA was found in tadpole liver, but not in tadpole choroid plexus. Thus, during evolution, synthesis of transthyretin in the liver of metamorphosing amphibians preceded that in the choroid plexus of reptiles, birds and mammals. It was previously observed that the protein most abundantly synthesized and secreted by the choroid plexus in adult amphibians is a lipocalin [Achen, M. G., Harms, P. J., Thomas, T., Richardson, S. J., Wettenhall, R. E. H. & Schreiber, G. (1992) J. Biol. Chem. 267, 23 170−23 174], in contrast to transthyretin being the most abundantly synthesized and secreted protein in the choroid plexus of mammals, birds and reptiles. Lipocalin mRNA was found in large amounts in tadpole choroid plexus, but not livers.
Association of His117 in the D2 protein of photosystem II with a chlorophyll that affects excitation‐energy transfer efficiency to the reaction center
Authors: Lince Michael T. , Vermaas Wim
Published: March 2003

His117 of the D2 protein of photosystem II (PS II) is a conserved residue in the second transmembrane region of the protein and has been suggested to bind chlorophyll. Nine site‐directed mutations were introduced at residue 117, using both photosystem I (PS I)‐containing and PS I‐less background strains of the cyanobacterium Synechocystis sp. PCC 6803. Of these nine, four (H117C, H117M, H117N, and H117T) were photoautotrophic in the PS I‐containing background. The other mutants (H117F, H117L, H117P, H117R, and H117Y) did not accumulate appreciable amounts of PS II in their thylakoids. The type of residues that can functionally replace His117 support the notion of His117 serving as a chlorophyll ligand. The properties of the H117N and H117T mutants were characterized in more detail. Whereas the properties of the H117N mutant were close to those of wild type, in the H117T mutant the 77‐K fluorescence emission spectrum shows a much smaller amplitude at 695 nm than expected on the basis of the amount of PS II that is present. Moreover, in H117T, the amount of light needed to half‐saturate O2‐evolution rates was twofold higher than in the control strain, and the variable fluorescence yield was quenched. However, O2 evolution rates at saturating light intensity and electron‐transport kinetics were normal in the mutant. Also, the radical accessory chlorophyll (Chl.es+.rb.eiZ.rb) formed by donation of an electron to the PS‐II reaction center could be generated normally by illumination at low temperature in the H117T mutant. We conclude that the chlorophyll associated with residue 117 of the D2 protein is important for efficient excitation transfer between the proximal antenna and the PS II reaction center. A possible mechanism involving a chlorophyll cation to explain the quenching in the H117T mutant is discussed.
Identification of an N‐hydroxyguanidine reducing activity of xanthine oxidase
Authors: Dambrova Maija , Uhlén Staffan , Welch Christopher J. , Wikberg Jarl E. S.
Published: March 2003

A guanoxabenz [1‐(2,6‐dichlorobenzylideneamino)‐3‐hydroxyguanidine; an N‐hydroxyguanidine] reducing enzymatic activity of rat spleen cytosol was investigated. By means of protein purification and N‐terminal amino acid sequencing, the reducing activity was shown to reside in xanthine oxidase. The action of the enzyme on guanoxabenz resulted in the formation of guanabenz [1‐(2,6‐dichlorobenzylideneamino)‐3‐guanidine]; the product formation could be monitored by HPLC and its identity was confirmed by NMR analysis. The reduction of guanoxabenz required xanthine or NADH as reducing substrates, while the process could be blocked by allopurinol, a selective inhibitor of xanthine oxidase. By using bovine milk xanthine oxidase, the guanoxabenz reducing activity of the enzyme was also verified. We conclude that guanoxabenz is a novel electron acceptor structure for xanthine oxidase.
Biosynthesis of the 3‐Acetyl and 131‐Oxo Groups of Bacteriochlorophyll a in the Facultative Aerobic Bacterium, Rhodovulum sulfidophilum
Authors: Porra Robert J. , Urzinger Markus , Winkler Johannes , Bubenzer Claudia , Scheer Hugo
Published: March 2003

Using 18O‐labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BChl a both aerobically in the dark and anaerobically in the light.

R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate 18O label from H218O into both the 131‐oxo and 3‐acetyl oxygens; in addition, the four carboxyl oxygens at C133 and C173 were labelled by H218O. Under aerobic conditions in the dark, the labelling of the 131‐oxo group by H218O was reduced indicating that 16O was being incorporated into this group from air.

R. sulfidophilum, grown in the dark under an atmosphere initially containing 50 %18O2 in Ar, possessed an oxygenase which incorporated 18O label from 18O2 specifically into the 131‐oxo group; under these conditions the acetyl and carboxyl groups remained unlabelled.

Thus, both an oxygenase and hydratase operate in R. sulfidophilum to form the 131‐oxo group of ring E of BChl a; the 3‐acetyl group oxygen, however, arises only from water via a hydratase.
The role of distal histidine in peroxidase activity of myoglobin
Authors: Khan Kishore Kuman , Mondal Madhu Sudan , Padhy Lakshmichand , Mitra Samaresh
Published: March 2003

The distal histidine has been proposed to play a crucial role in the reaction of peroxidases with hydroperoxide. Myoglobin (Mb), due to its close similarity with peroxidases, also reacts with various peroxides. The effect of mutation of the distal histidine on the peroxidase activity of Mb has been investigated by stopped‐flow kinetics of the reaction of hydrogen peroxide with wild‐type Mb and [Gly64]Mb. The results indicate kinetic and mechanistic differences in the formation of peroxide compounds from these two forms of Mb. The rate of reaction of H2O2 with the wild‐type Mb decreased 8−9‐fold on mutation of the distal histidine to glycine ([Gly64]Mb). A second slow phase was observed for the reaction of H2O2 with [Gly64]Mb, but was not observed in the corresponding reaction with wild‐type Mb. It is suggested that the decrease in the rate of the reaction on mutation is due to the absence of a general acid‐base catalyst. The effect of pH on the rate of reaction of H2O2 with wild‐type Mb and [Gly64]Mb is contrasting. While the rate of the M6 reaction decreased for wild‐type Mb at higher pH, probably due to the acid‐alkaline transition of Mb at higher pH, the rate of reaction was found to increase at higher pH for mutant Mb. The increase in the rate of the reaction is suggested to be due to an increase in the ionization of H2O2 at higher pH, the rate‐determining step being the formation of the intermediate Fe‐O‐O‐H complex and not the subsequent step of oxo‐ferryl complex formation. The thermodynamic parameters calculated from the temperature‐dependent study showed that the enthalpy of binding of H2O2with Mb is positive, indicating that the process is endothermic. The apparent energy of activation of the reaction of H2O2with Mb was found to be higher than that of peroxidases, suggesting that this may be oue of the reasons for the slower rate of the reaction of H2O2with Mb compared with peroxidases.
The deletion of the C‐terminal tail and addition of an endoplasmic reticulum targeting signal to Alzheimer's amyloid precursor protein change its localization, secretion, and intracellular proteolysis
Authors: Kouchi Zen , Kinouchi Tadatoshi , Sorimachi Hiroyuki , Ishiura Shoichi , Suzuki Koichi
Published: March 2003

The metabolic pathway of Alzheimer's amyloid precursor protein (APP) involves restricted intracellular proteolysis by secretases, which leads to the secretion of the N‐terminal soluble APP (sAPP) and the generation of a cell‐associated C‐terminal fragment. The precise cellular sites at which these processes occur remain unknown. In this report, we describe the route of APP sorting and the processing site using novel systems with and without sorting signals on the APP molecule. One system involves the replacement of the C‐terminal ten amino acids of APP with Adenoviral E19 protein containing an endoplasmic reticulum (ER) retrieval signal (APPE19); the other involves deleting the last ten amino acids correponding to the replaced site (APPdeltaC10). APPE19 localized mainly within the cis/medial Golgi compartment and exclusively suppresses the secretion of APP. In contrast, deletion of the C‐terminal tail promotes sAPP secretion by a constitutive secretion pathway. Metabolic labeling followed by immunoprecipitation with anti‐APP antibody revealed that APPE19 is rapidly degraded within 30 min and that the subsequent intracellular turnover rate is decreased with 40 % of the protein retained within the cells even after a chase period a 3 h. In contrast, APPdeltaC10 is rapidly eliminated from the intracellular compartments and secreted into the culture medium. The surface internalization and recycling processes of this protein are relatively impaired compared with wild‐type APP. The ratios of the levels of production to secretion of sAPPα, the N‐terminal, soluble APP fragment released by α‐secretase, are proportional to the secretion efficiencies among APP species, suggesting the localization of α‐secretase within a compartment late in the constitutive secretion pathway. These secretion mutants which utilize ER targeting signals are useful tools for analyzing the location of secretases and the intracellular degradation system within a constitutive secretion pathway such as ER quality control.
Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast
Authors: Jouannic Nathalie , Lepetit Marc , Vergnolle Chantal , Cantrel Catherine , Gardies Anne‐Marie , Kader Jean‐Claude , Arondel Vincent
Published: March 2003

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol‐transfer protein (Sec14psc) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so‐called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14psc protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14ts mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14ts mutant at the restrictive temperature of 37 +C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol‐transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14ts mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5 % similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.
Sporulation‐specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p
Authors: Bogengruber Edith , Eichberger Thomas , Briza Peter , Dawes Ian W. , Breitenbach Michael , Schricker Roland
Published: March 2003

Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation. Transcription of these two divergently arranged genes is controlled by a common 900‐bp intergenic region. Random mutagenesis of this promoter and tests with appropriate reporter constructs identified an 11‐bp cis‐acting palindromic sequence, DIT repressor element (DRE), as a major negative regulatory site during vegetative growth. Repression is exerted by DRE in conjunction with a mid‐sporulation element (MSE)‐like sequence situated 26 bp away. These sequence elements are both contained within the 76‐bp negative regulatory element (NRE) defined previously [Friesen H., Hepworth, S. R. & Segall, J. (1997) Mol. Cell. Biol. 17, 123−134]. The activated form of Rim101p, a transcriptional inducer of the early meiotic gene IME1, enhances expression from the DIT1 promoter both in vegetative and sporulating cells. Activation by Rim101p does not seem to involve binding of Rim101p at either of the two cis‐acting sites described here, since reporter constructs with both elements or most of the NRE deleted could still be activated by the short form of Rim101p.
Neutral core oligosaccharides of bovine submaxillary mucin
Authors: Mårtensson Stig , Levery Steven B. , Fang Tammy T. , Bendiak Brad
Published: March 2003

Oligosaccharide alditols were released from bovine submaxillary mucin by alkaline borohydride treatment. The fractions containing smaller neutral oligosaccharides were separated by HPLC, to give, in the mono‐, di‐ and trisaccharide‐alditol sizes, 19 different structures, in addition to three fucosylated tetrasaccharide alditols. Molecules were identified by NMR spectroscopy, electrospray‐mass‐spectrometry−mass‐spectrometry, permethylation analyses, and highly selective Pb(OAc)4 oxidation at −30 +C, followed by borohydride reduction. Pb(OAc)4 oxidation was found to be generally applicable in identifying the branch position(s) of substitutions for all core structures. Among the isolated oligosaccharide alditols were structures not previously reported, including 12 structures not reported from bovine submaxillary mucin, and four structures (three core structures) not found in the Carbbank database.
Biophysical characterisation of lysozyme binding to LPS Re and lipid A
Authors: Brandenburg Klaus , Koch Michel H. J. , Seydel Ulrich
Published: March 2003

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the ’endotoxic principle' of LPS, was investigated using biophysical techniques. The β  α gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier‐transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X‐ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl‐chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stochiometry of endotoxin and lysozyme is a 3 : 1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A‐phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly α helix to more unordered structures takes place, while the residual β‐sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.
Structural determination of sulfated tetrasaccharides and hexasaccharides containing a rare disaccharide sequence, ‐3GalNAc(4,6‐disulfate)β1‐4IdoAα1‐, isolated from porcine intestinal dermatan sulfate
Authors: Yamada Shuhei , Yamane Yukari , Sakamoto Kaori , Tsuda Hiromi , Sugahara Kazuyuki
Published: March 2003

In the course of structural studies on sulfated oligosaccharides isolated from porcine intestinal heparin after extensive digestion with Flavobacterium heparinase, we isolated several heparitinase‐resistant unsaturated oligosaccharides. Amino sugar analysis of these oligosaccharides indicated that they contained galactosamine residues but no glucosamine residues. They were sensitive to chondroitinase ABC but resistant to chondroitinase AC‐II, and therefore derived from dermatan sulfate, which was presumably contained as a minor component in the starting heparin preparation. The structures of these oligosaccharides were characterized by enzymatic digestions in conjunction with HPLC analysis of the digests and by one‐dimensional and two‐dimensional 500‐MHz 1H‐NMR spectroscopy. Structures of two tetrasaccharides and two hexasaccharides were determined as ΔHexAα1‐3GalNAc(4S)β1‐4IdoAα1‐3GalNAc(4S), ΔHexAα1‐3GalNAc(4S,6S)β1‐4IdoAα1‐3GalNAc(4S), ΔHexAα1‐3GalNAc(4S)β1‐4IdoAα1‐3GalNAc(4S)β1‐4IdoAα1‐3GalNAc(4S), and ΔHexAα1‐3GalNAc(4S)β1‐4IdoAα1‐3GalNAc(4S,6S)β1‐4IdoAα1‐3GalNAc(4S), where ΔHexA, IdoA, GalNAc, 4S and 6S represent 4‐deoxy‐α‐
L‐threo‐hex‐4‐enepyranosyluronic acid,
L‐iduronic acid, N‐acetyl‐
D‐galactosamine, 4‐O‐sulfate and 6‐O‐sulfate, respectively. The latter three compounds have never been reported as discrete structures. Since the four isolated oligosaccharides contained an unsaturated uronic acid residue at the nonreducing terminus, they appear to have been generated by eliminative cleavage by the action of Flavobacterium chondroitinase that was probably present as a minor contaminant in the Flavobacterium heparinase preparation used. Two out of the four oligosaccharides shared the rare disulfated disaccharide sequence, ‐3GalNAc(4S,6S)β1‐4IdoAα1. These oligosaccharides will be useful as authentic reference compounds for microanalyzing biologically active domains of dermatan sulfate.