bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction with phospholipids of a membrane thiol peptidase that is essential for the signal transduction of mating pheromone in Rhodosporidium toruloides
Authors: JEONG Yong Kee , MIYAKAWA Tokichi , IMABAYASHI Akemi , TSUCHIYA Eiko , FUKUI Sakuzo
Published: March 2005

Interaction with phospolipids of a membrane thiol peptidase [referred to as trigger peptidase (TPase), T. Miyakawa et al. (1987) J. Bacteriol. 169, 1626–1631] that plays a key role in the signalling of a lipopeptidyl mating pheromone at the cell surface of pheromone‐target cell (mating type a) of Rhodosporidium toruloides was studied. The activity of highly purified TPase which requires phospholipids was restored by reconstitution of the enzyme into liposomes prepared with phospholipids extracted from the yeast cell. The presence of Ca2+ was essential for both the reconstitution process and the catalytic reaction of TPase. Triton X‐100 mixed micelles containing phospholipids also activated the enzyme. The specificity and stoichiometry of activation by phospholipids was investigated by determination of TPase in the presence of mixed micelles that contained defined classes and numbers of phospholipid molecules in the Triton X‐100 micelles. It was demonstrated that TPase is activated by mixed micelles containing 2–6 molecules of phosphatidylserine or phosphatidylethanolamine. Other phospholipids of the membranes of this organism, such as phosphatidylcholine and phosphatidylglycerol, had little effect on activation, indicating that the amino group of the phospholipids may be required for the function of TPase. Direct evidence for the interaction of TPase and Triton X‐100/phosphatidylserine mixed micelles was obtained by molecular sieve chromatography on Sephacryl S‐200. These data established that a phospholipid bilayer is not a requirement for TPase activation, and that the purified enzyme can be activated by a relatively small number of phospholipid molecules of specific classes.
The mitochondrial genome of the fission yeast, Schizosaccharomyces pombe
Authors: LANG B. Franz , CEDERGREN Robert , GRAY Michael W.
Published: March 2005

The DNA sequence of the mitochondrial large subunit (LSU) rRNA gene of Schizosaccharomyces pombe has been determined. In the direction of transcription, this gene is located between the gene coding for subunit II of cytochrome oxidase and a cluster of three tRNA genes. Both the 5′ and 3′ ends of the LSU rRNA have been mapped precisely: whereas the 5′ end can be assigned unambiguously to a single nucleotide position, multiple 3′ ends occur within a run of eight U residues. Based on these results, the S. pombe LSU rRNA is between 2818 and 2826 nucleotides long. A sequence motif immediately upstream of the 5′ end of the gene resembles that of the mitochondrial prømoter motif of Saccharomyces cerevisiae; however, the sequence at the 3′ end of the gene is not similar to any of the motifs implicated as processing signals in other mitochondrial systems. Unlike its counterparts in S. cerevisiae and Aspergillus nidulans, the mitochondrial LSU rRNA gene of S. pombe does not contain an intron. Comparison of potential secondary structure among the three fungal mitochondrial and Escherichia coli LSU rRNAs has defined a common secondary structure core, held together by long‐range hydrogen‐bonding interactions. A 5.8S‐like structure is present within the 5′‐terminal region of all three fungal mitochondrial LSU rRNAs; in contrast, no 4.5S‐like structure is evident at the 3′ end of these molecules. An evolutionary evaluation of highly conserved regions of a small set of LSU rRNA sequences suggests that S. pombe mitochondria diverged from a mitochondrial proto‐fungal branch earlier than either A. nidulans or S. cerevisiae mitochondria. This result, considered in conjunction with the patterns of genome organization and codon usage in fungal mitochondria, points to a slower evolutionary clock speed in the mitochondrial genome of S. pombe.
Amino‐acid sequence of the cytochrome‐b5‐like heme‐binding domain from Hansenula anomala flavocytochrome b2
Authors: HAUMONT Pierre‐Yves , THOMAS Marie‐Antoinette , LABEYRIE Françoise , LEDERER Florence
Published: March 2005

Flavocytochrome b2 (L‐lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme‐binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539–2543]. We report here the amino acid sequence of the heme‐binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme‐binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three‐dimensional structure of S. cerevisiae flavocytochrome b2 [Z‐x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629–2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563–568].
Convulsant/barbiturate activity on the soluble γ‐aminobutyric acid – benzodiazepine receptor complex
Authors: KING Ronnie G. , NIELSEN Mogens , STAUBER Greg B. , OLSEN Richard W.
Published: March 2005

Cage convulsant t‐butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3‐[(3‐cholamidopropyl)‐dimethylammonio]propane sulfonate (Chaps) and shown to co‐purify with the benzodiazepine –γ‐aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X‐100, or by addition of Triton X‐100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA – benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X‐100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3 – 5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.
Formation of lipoxin B by the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate
Authors: KÜHN Hartmut , WIESNER Rainer , ALDER Lutz , FITZSIMMONS Brian J. , ROKACH Joshua , BRASH Alan R.
Published: March 2005

The pure reticulocyte lipoxygenase converts 15Ls‐hydroxy‐5,8,11,13(Z,Z,Z,E)‐icosatetraenoic acid (15Ls‐HETE) methylester to a complex mixture of products containing 5Ds,14LR,15Ls‐trihydro(pero)xy‐6E,8Z,10E,12E‐icosatetraenoate methyl ester (lipoxin B methyl ester), 5Ds,15Ls‐DiH(P)ETE methyl ester and four 8,15Ls‐DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy‐icosatetraenoic acid].

After a short incubation period (15 min) 5Ds,15Ls‐DiH(P)ETE methyl ester was found to be the main product, whereas after a 3‐h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400‐MHz 1H‐NMR.

Atmospheric oxygen was incorporated at carbon‐5 and carbon‐14 into the major product. 5Ds,15Ls‐DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15Ls‐HETE and 5Ds,15Ls‐DiHETE.

The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.
One‐ and two‐dimensional 1H‐NMR investigations of two 19‐base‐pair analogues of the tet operator
Authors: STOLARSKI Ryszard , BUCK Friedrich , FERA Brian , RÜTERJANS Heinrich
Published: March 2005

Two 19‐base‐pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline‐resistance (tet) determinant, have been studied by 1H‐NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one‐dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two‐dimensional NOE spectroscopy in H2O. Two‐dimensional phase‐sensitive NOE and magnitude‐correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5′, H5” protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B‐DNA, show irregularities in the mismatch areas.
Lipid interaction of diphtheria toxin and mutants with altered fragment B
Authors: PAPINI Emanuele , COLONNA Raffaele , CUSINATO Federico , MONTECUCCO Cesare , TOMASI Maurizio , RAPPUOLI Rino
Published: March 2005

The interaction of diphtheria toxin and its cross‐reacting mutants crm 45, 228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing.

The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5–6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.
Lipid interaction of diphtheria toxin and mutants with altered fragment B
Authors: PAPINI Emanuele , SCHIAVO Giampietro , TOMASI Maurizio , COLOMBATTI Marco , RAPPUOLI Rino , MONTECUCCO Cesare
Published: March 2005

The membrane insertion of diphtheria toxin and of its B chain mutants crm 45, crm 228 and crm 1001 has been followed by hydrophobic photolabelling with photoactivatable phosphatidylcholine analogues. It was found that diphtheria toxin binds to the lipid bilayer surface at neutral pH while at low pH both its A and B chains also interact with the hydrocarbon chains of phospholipids. The pH dependence of photolabelling of the two protomers is different: the pKa of fragment B is around 5.9 while that of fragment A is around 5.2. The latter value correlates with the pH of half‐maximal intoxication of cells incubated with the toxin in acidic mediums. These results suggest that fragment B penetrates into the bilayer first and assists the insertion of fragment A and that the lipid insertion of fragment B is not the rate‐controlling step in the process of membrane translocation of diphtheria toxin.

crm 45 behaves as diphtheria toxin in the photolabelling assay but, nonetheless, it is found to be three orders of magnitude less toxic than diphtheria toxin on acid‐treated cells, suggesting that the 12‐kDa COOH‐terminal segment of diphtheria toxin is important not only for its binding to the cell receptor but also for the membrane translocation of the toxin.

It is suggested that crm 1001 is non‐toxic because of a defect in its membrane translocation which occurs at a lower extent and at a lower pH than that of the native toxin; as a consequence crm 1001 may be unable to escape from the endosome lumen into the cytoplasm before the fusion of the endosome with lysosomes.
Binding of brain spectrin to the 70‐kDa neurofilament subunit protein
Authors: FRAPPIER Thierry , REGNOUF Françoise , PRADEL Louise Anne
Published: March 2005

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70‐kDa neurofilament subunit (NF‐L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin‐binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was ‘blotted' with 125I‐fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF‐L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF‐L was reversible and concentration‐dependent. The ability of the pure NF‐L and GFP to form filaments was used to quantify their association with fodrin.

The binding of fodrin to reassembled NF‐L was saturable with a stoichiometry of 1 mol fodrin bound/50±10 mol NF‐L and an apparent dissociation constant Kd= 4.3 × 10−7 M.

The binding involved the N‐terminal domain of the polypeptide chain derived from the [2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine] cleavage of NF‐L.

Binding occurred optimally at physiological pH (6.8‐7.2) and salt concentrations (50 mM).

Interestingly, calmodulin, a Ca2+‐binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF‐L, at Ca2+ physiological concentrations.

The binding of fodrin to pure neurofilaments was not affected by the presence of the 200‐kDa (NF‐H) and the 160‐kDa (NF‐M) subunits. The apparent dissociation constant for the binding of fodrin to NF‐L in the pure NF was 1.0 × 10−6 M with 1 mol fodrin bound/80 ± 10 mol NF‐L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 × 10−7 M and the maximum binding was 1 mol fodrin/55 ± 10 mol GFP.
Properties of the Na+‐dependent Cl−/HCO−3 exchange system in U937 human leukemic cells
Authors: LADOUX Annie , KRAWICE Irène , CRAGOE Edward J. , ABITA Jean Pierre , FRELIN Christian
Published: March 2005

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride‐sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl− influx and efflux experiments.

Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi= 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate‐stimulated uptake pathway for Na+ which was inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate. The bicarbonate‐dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl− and increased by removal of external Cl−. 36Cl− efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl− at a cell pHi of 6.3. Finally a 36Cl− uptake pathway was documented. It was inhibited by 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (K0.5= 10 μM) and bicarbonate (K0.5= 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+‐independent Cl−/HCO−3 exchange system could be found. The Na+‐dependent Cl−/HCO−3 exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate‐induced swelling of astroglia and of the Li(Na)CO−3/Cl‐ exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.
Structure of the capsular polysaccharide of Haemophilus pleuropneumoniae serotype 5
Authors: ALTMAN Eleonora , BRISSON Jean‐Robert , PERRY Malcolm B.
Published: March 2005

The capsular polysaccharide of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 (ATCC 33377) was found to be a linear type polysaccharide of a repeating disaccharide unit composed of 2‐acetamido‐2‐deoxy‐d‐glucose and 3‐deoxy‐d‐manno‐2‐octulosonic acid (dOclA). By composition analysis, methylation, partial hydrolysis and 1H and 13C nuclear magnetic resonance studies, it was concluded that the capsular polysaccharide is a high‐molecular‐mass unbranched polymer having the structure:

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Prostaglandin release but not superoxide production by rat Kupffer cells stimulated in vitro depends on Na+/H+ exchange
Authors: DIETER Peter , SCHULZE‐SPECKING Agnes , KARCK Ullrich , DECKER Karl
Published: March 2005

The release of the prostaglandins E2 and D2, induced by zymosan and phorbol ester in cultured rat Kupffer cells, was found to depend on the extracellular concentration of Na+. Eicosanoid formation following the administration of the Ca2+ ionophore A23187 or of arachidonic acid, however, did not require the presence of sodium ions in the medium. A half‐maximal rate of prostaglandin release by zymosan‐treated Kupffer cells was obtained between 4 mM and 5 mM Na+; and a Na+ concentration of ≥ 30 mM was required to maximally stimulate prostaglandin E2 and D2 formation in the cultured liver macrophages. In contrast, the superoxide production following the administration of zymosan or of phorbol ester was quite independent of extracellular Na+.

The zymosan and phorbol‐ester‐stimulated release of prostaglandins E2 and D2 was inhibited by amiloride. Artificial intracellular alkalization enhanced the prostanoid production of unstimulated and of zymosan‐stimulated cells whereas artificial intracellular acidification inhibited the zymosan‐elicited prostaglandin synthesis. In contrast, the superoxide formation was independent of the pH changes. The data presented here suggest that the prostaglandin production elicited by zymosan or phorbol ester in cultured rat Kupffer cells requires an activated Na+/H+ exchange.
The function of the human factor V carbohydrate moiety in blood coagulation
Authors: BRUIN Taco , STURK Augueste , TEN CATE Jan W. , CATH Marieke
Published: March 2005

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5–2‐fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin‐induced activation, whereas deglycosylated factor V activation was impaired. Lectin‐blot experiments with sialic‐acid‐specific Limax flavus agglutinin (LFA), galactose‐specific Ricinus communis agglutinin (RCA‐II) and mannose‐specific concanavalin A on thrombin‐induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1 F2 contained terminal galactose residues. LFA and RCA‐II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.
Primary structure of the glycans of porcine pancreatic lipase
Authors: FOURNET Bernard , LEROY Yves , MONTREUIL Jean , DĚCARO Josiane , ROVERY Mireille , van KUIK J. Albert , VLIEGENTHART Johannes F. G.
Published: March 2005

The glycan primary structure of the main glycopeptide fraction obtained by pronase and carboxypeptidase A digestions of porcine pancreatic lipase has been investigated by 500‐MHz1H‐NMR spectroscopy and methylation analysis. The results demonstrate that the glycopeptide fraction was a mixture containing the following structures:

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Relations between plasma membrane and lysosomal membrane
Authors: DRAYE Jean‐Pierre , QUINTART Joël , COURTOY Pierre J. , BAUDHUIN Pierre
Published: March 2005

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4°C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37°C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell‐associated labels) was rapidly released (half‐life < 1 h), and most label (65%) was released slowly (half‐life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37°C, the patterns of 125I‐polypeptides quantified by autoradiography after SDS‐PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides.

The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2 – 4% of cell‐associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H‐labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell‐associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.
Metabolism of the vitamin D3 derivative (24R)‐hydroxycalcidiol by human promyelocytic leukemia cells (HL‐60)
Authors: ISHIZUKA Seiichi , MATSUI Toshimitsu , NAKAO Yoshinobu , FUJITA Takuo , REICHEL Helmut , NORMAN Anthony W.
Published: March 2005

Human promyelocytic leukemia cells (HL‐60 cells) incubated with (24R)‐hydroxy[26,27‐methyl‐3H]calcidiol (0.2 μCi) or non‐radioactive (24R)‐hydroxycalcidiol (370 μg) produced significant quantities of two new vitamin D3 (calciol) metabolites. The metabolites were isolated from HL‐60 cell culture media by methanol/chloroform extraction and a series of chromatographic procedures. The two new metabolites were identified as (5Z)‐ and (5E)‐(24R)‐19‐nor‐10‐oxo‐24‐hydroxycalcidiol by HPLC analysis, ultraviolet absorption spectrophotometry, mass spectrometry and Fourier‐transfrom infrared spectrophotometry. According to the isolation and purification procedures, the total amounts of 3.04 μg (5Z)‐(24R)‐19‐nor‐10‐oxo‐24‐hydroxycalcidiol (λmax= 310 nm, ɛ= 17070 M−1 cm−1) and 8.89 μg (5E)‐(24R)‐19‐nor‐10‐oxo‐24‐hydroxycalcidiol (λmax= 312 nm, e= 24500 M−1 cm−1) were calculated, assuming an M1 of 418. The activity of 19‐nor‐10‐oxo‐(24R)‐hydroxycalcidiol to promote HL‐60 cell differentiation was higher than the activity of the precursor (24R)‐hydroxycalcidiol suggesting a possible biological action of this metabolite in HL‐60 cells.
Purification and characterization of peptide‐elongation factor 2 (aEF‐2) from an extremely halophilic archaebacterium Halobacterium halobium
Authors: SARUYAMA Haruo , SASAKI Shoji
Published: March 2005

A procedure is described for the purification of the archaebacterial peptide‐elongation factor 2 (aEF‐2) from an extremely halophilic archaebacterium Halobacterium halobium. The enrichment was about 530‐fold, the obtained preparation practically homogeneous as judged by SDS‐PAGE. The poly(U)‐dependent poly(Phe) synthesis was completely dependent on aEF‐2 in the presence of partially purified aEF‐1, and the activity was equivalent to a poly(Phe)‐synthesizing system containing unfractionated S‐100 enzymes, aEF‐2 consists of a single peptide with a relative molecular mass of 125000 ± 3000 and 100000 ± 3000 as determined by SDS‐PAGE and gel filtration on Sephadex G‐200 respectively. The isoelectric point was 5.7. The amino acid composition analysis indicated the predominance of acidic amino acids (aspartic acid and glutamic acid) and the low content of hydrophobic amino acid (phenylalanine) as compared with those of eukaryotes and prokaryotes. The factor was stable in a pH range from 6 to 8. 2‐Mercaptoethanol and GTP but not GDP markedly protected aEF‐2 from heat denaturation at 52 °C. aEF‐2 became inactivated and insensitive to ADP‐ribosylation by diphtheria toxin at low ionic strength but could be renatured by increasing ionic strength. Obviously higher concentrations of salts contribute to the conformational stability of aEF‐2.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: TOMME Peter , VAN TILBEURGH Herman , PETTERSSON Göran , VAN DAMME Jozef , VANDEKERCKHOVE Joel , KNOWLES Jonathan , TEERI Tuula , CLAEYSSENS Marc
Published: March 2005

Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).

By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43–52].

The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N‐terminal region contributes in the binding onto both cellulose types; the homologous C‐terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.
Biosynthesis of monoterpene hydrocarbons by isolated chromoplasts from daffodil flowers
Authors: METTAL Ursula , BOLAND Wilhelm , BEYER Peter , KLEINIG Hans
Published: March 2005

Incubation studies using [1‐14C]isopentenyl diphosphate and [1‐2H2]isopentenyl diphosphate as substrates revealed that isolated chromoplasts from flowers of Narcissus pseudonarcissus L. are able to synthesize monoterpene hydrocarbons and linalool in high yields. The enzymes involved are soluble in the chromoplast stroma. It is hypothesized that in the plant cell plastids are the site of monoterpene biosynthesis, whereas the formation of sesquiterpenes may be restricted to the cytoplasm/endoplasmic reticulum.
Fo part of the ATP synthase from Escherichia coli
Authors: STEFFENS Karl , HOPPE Jürgen , ALTENDORF Karlheinz
Published: March 2005

Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b. Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding [Schneider, E. and Altendorf, K. (1985) EMBO J. 4, 515–5181. The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3‐(trifluoromethyl)‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence. In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID‐labelled membrane vesicles of Escherichia coli [Hoppe et al. (1984) Biochemistry 23, 5610–56161. In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified. The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits.
Purification and characterization of a 22‐kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger
Authors: THIEL Gerald , SCHMIDT Wolfgang E. , MEYER Helmut E. , SÖLING Hans‐Dieter
Published: March 2005

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345–352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. It could be extracted from the endoplasmic reticulum membrane only with Triton X‐100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X‐100, removal of the detergent by acetone precipitation, extraction of water‐soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic PI (> 8.7). For determination of the amino acid composition of protein III and for sequencing of its amino‐terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed‐phase HPLC. Protein III could be phosphorylated in vitro by the catalytic subunit of the CAMP‐dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys‐Leu‐Ser(P)‐Glu‐Ala‐Asp‐Asn‐Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with CAMP‐dependent protein kinase. The first 41 N‐terminal residues of protein III were sequenced. So far no sequence homology with other known peptides or proteins could be found.
Elicitor‐induced tyrosine decarboxylase in berberine‐synthesizing suspension cultures of Thalictrum rugosum
Authors: GÜGLER Karl , FUNK Christoph , BRODELIUS Peter
Published: March 2005

Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline‐derived alkaloids.
Identification and primary structure of the cardiolipin‐binding domain of mitochondrial creatine kinase
Authors: CHENEVAL Dominique , CARAFOLI Ernesto
Published: March 2005

It was recently shown that the mitochondrial isozmye of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839–3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin‐containing liposomes and has thus been identified as the cardiolipin‐binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N‐terminal region of the protein. The binding of the fragment of cardiolipin‐containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate‐oxidized (di‐aldehyde) ATP and has thus been identified as the ATP‐binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.
Insect immunity
Authors: DIMARCQ Jean‐Luc , KEPPI Elisabeth , DUNBAR Bryan , LAMBERT Jean , REICHHART Jean‐Marc , HOFFMANN Danièle , RANKINE Susan M. , FOTHERGILL John E. , HOFFMANN J. A.
Published: March 2005

Injury or injection of live bacteria into third instar larvae of the dipteran insect Phormia terranovae results in the appearance in the haemolymph of at least five groups of heat‐stable, more or less basic peptides with antibacterial activity against Escherichia coli. Three of these peptides have been purified. The amino acid sequence has been completely established for one of these and partially (first 40 residues from the N‐terminus) for the two others. The sequences show marked homologies indicating that the three peptides belong to a common family. They are not related to other known antibacterial peptides from insects [lysozymes, cecropins (including sarcotoxin I) and attacins]. We propose the name of diptericins for this new family of antibiotic molecules.
Conformational analysis of the dinucleotides 5′‐methylphospho‐N6‐dimethyladenylyl‐uridine (mpm62A‐U) and 5′methylphospho‐uridylyl‐N6‐dimethyladenosine (mpU‐m62A) and of the trinucleotide U‐m62A‐U
Authors: van den HOOGEN Yvonne T. , LANKHORST Peter P. , GIJSMAN Pieter , HARTEL Arnold J. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

NMR and CD studies were carried out on the dinucleotides 5′‐methylphospho‐N6‐dimethyladenylyl‐uridine (mpm62‐U) and 5′‐methylphospho‐uridylyl‐N6‐dimethyladenosine (mpU‐m62A) and on the trinucleotide U‐m62A‐U. A detailed comparison is given of the conformational features of mpm62A‐U and mpU‐m62A with the corresponding 5′‐nonphosphorylated dinucleotides m62A‐U and U‐m62A, respectively. The behaviour of the trinucleotide U‐m62A‐U is compared with the properties of the constituent dinucleotides U‐m62A and mpm62A‐U.

Chemical‐shift and CD data were used to determine the amount of stacking interactions. For each compound NMR spectra were recorded at two or three sample concentrations in order to separate intermolecular and intramolecular base‐base interactions. The coupling constants of the ribose ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles β, γ and ɛ are presented.

The combined data indicate a strong similarity between mpm62A‐U and m62A‐U both in degree and in mode of stacking. In contrast, the existence of different types of stacking interactions in mpU‐m62A and U‐m62A is suggested in order to explain the NMR and CD data. It is concluded that dinucleoside bisphosphates serve better as a model for the behaviour of trinucleotides than dinucleoside monophosphates.

The trinucleotide U‐m62A‐U adopts a regular single‐stranded stacked RNA structure with preference for N‐type ribose and γ+ and βt backbone torsion angles. The difference in behaviour between the U‐m62A‐ part of U‐m62A‐U and the dimer U‐m62A is seen as a typical example of conformational transmission.
The secondary structure of the toxin ATX Ia from Anemonia sulcata in aqueous solution determined on the basis of complete sequence‐specific 1H‐NMR assignments
Authors: WIDMER Hans , WAGNER Gerhard , SCHWEITZ Hugues , LAZDUNSKI Michel , WÜTHRICH Kurt
Published: March 2005

The toxin preparations ATX I, ATX Ia and ATX Ib from Anemonia sulcata were investigated by proton nuclear magnetic resonance (NMR). High‐resolution phase‐sensitive two‐dimensional NMR experiments were used to monitor the separation by high‐performance liquid chromatography of the two isoproteins ATX Ia and ATX Ib. For ATX Ia complete sequence‐specific resonance assignments were obtained and the secondary structure was determined. To obtain the NMR assignments we used a variant of the sequential assignment technique which relied extensively on cross‐peak fine‐structure analysis in phase‐sensitive spectra, using spectrum simulations based on density matrix calculations with the program SPHINX. These procedures, which resulted in extensive amino acid spin system identifications prior to the sequential assignments, should be generally applicable for small proteins with relatively narrow 1H‐NMR lines. The secondary structure of ATX I includes a β sheet consisting of four strands. No evidence was found for the presence of regular helical segments. The four β strands are connected by two extended loops and a tight turn, for which further characterization has to await the complete determination of the three‐dimensional structure.
Inhibition of human glutathione reductase by the nitrosourea drugs 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea and 1‐(2‐chloroethyl)‐3‐(2‐hydroxyethyl)‐1‐nitrosourea
Authors: KARPLUS P. Andrew , KRAUTH‐SIEGEL R. Luise , SCHIRMER R. Heiner , SCHULZ Georg E.
Published: March 2005

Glutathione reductase from human erythrocytes was inhibited by incubation with the drugs 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) and 1‐(2‐chloroethyl)‐3‐(2‐hydroxyethyl)‐1‐nitrosourea (HeCNU) under quasi‐physiological conditions. For reference purposes, iodoacetamide was used for inactivating alkylation of the enzyme. In each case the modified glutathione reductase was crystallized and its structure determined. These analyses showed that in all experiments the enzyme had reacted at the distal sulfur, that is at the thiol of Cys‐58, and virtually nowhere else in the visible structure. The electron density of the HeCNU derivative at 0.3 nm resolution is consistent with a 2‐hydroxyethyl group. This alkyl moiety has recently been identified by chemical analysis [Schirmer, R.H., Schöllhammer, T., Eisenbrand, G. and Krauth‐Siegel, R.L. (1987) Free Radical Res. Commun. 3, 3–12]. The 0.2 nm resolution electron‐density map of the BCNU‐derivatized enzyme cannot be explained by a 2‐hydroxyethyl group. Instead the modification appears as a carbamoyl moiety containing at least five non‐hydrogen atoms. In this derivative the distal cysteine is forced into an unusual conformation.
Two different adenylyl cyclases in brain distinguished by monoclonal antibodies
Authors: MOLLNER Stefan , PFEUFFER Thomas
Published: March 2005

Four monoclonal antibodies, raised against the 115‐kDa adenylyl cyclase from bovine brain [Pfeuffer, E. et al. (1985) EMBO J. 4, 3675–3679] have been selected and designated BBC‐1 to BBC‐4. BBC‐1 and BBC‐3 are highly specific for the 115‐kDa enzyme from bovine brain. The two other antibodies, BBC‐2 and BBC‐4, recognize an additional 150‐kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150‐kDa species is detected by the crossreacting antibodies BBC‐2 and BBC‐4.The two adenylyl cyclases from brain can be separated by calmodulin‐Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.
Reversal of snake neurotoxin binding to mammalian acetylcholine receptor by specific antiserum
Authors: GATINEAU Eric , LEE Chen Yuan , FROMAGEOT Pierre , MENEZ André
Published: March 2005

Snake curaremimetic toxins are known to bind to the nicotinic acetylcholine receptor (AcChoR)[Changeux et al. (1970) Proc. Natl Acad. Sci. USA, 67, 1241–1247], thus blocking neuromuscular transmission, and producing respiratory failure in mammals. In the present paper we show that the toxic effects of Naja nigricollis toxin α to mammals can be efficiently reversed by toxin‐α‐specific antibodies. In vivo we observed that return to normal breathing in toxin‐α‐intoxicated and ventilated rats was 12 times faster after injection of specific antiserum or monoclonal antibody (Mα1) as compared with control animals. Ex vivo we observed that return to normal contraction of a toxin‐α‐blocked phrenic nerve‐hemidiaphragm preparation was 14 times more rapid after treatment with specific antiserum than after washings. In vitro we observed that antibodies accelerated the reversal of binding of [3H]toxin α to AcChoR prepared from rat diaphragm. The observation made in vitro furthermore indicates that antibodies are capable of destabilizing the [3H]toxin‐AcChoR complex. A similar destabilization phenomenon occurs also in vivo, as inferred from measurements of receptor occupancy by [3H]toxin α in diaphragm of anaesthetized rats in the presence or absence of antibodies. The property of antibodies to reverse neurotoxin binding to AcChoR may be considered as a critical test for evaluation of the quality of a neurotoxin‐specific antisera.
Primary structure of neutral oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500‐MHz 1H‐NMR spectroscopy and quantitative sugar analysis
Authors: BREG Jan , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G. , KLEIN André , LAMBLIN Geneviève , ROUSSEL Philippe
Published: March 2005

A pool of neutral carbohydrate chains was prepared from respiratory mucins of a patient suffering from bronchiectasis. Fractionation by HPLC led to 35 smaller‐size oligosaccharide‐alditols; the structure of 16 oligosaccharide‐alditols with core type 1 or type 2 has been established (Klein, A., Lamblin, G., Lhermitte, M., Roussel, P., Breg, J., Van Halbeek, H. & Vliegenthart, J. F. G., preceding paper in this journal).

In this second part, we identified 19 oligosaccharide‐alditols possessing core types 3 and 4. Nine of the structures (1, 5, 9, 6, 10b, 13, 19, 15b and 18.1) have been described previously to be present in cystic fibrosis mucins [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J. G. H. M., Van Halbeek, H. & Vliegenthart, J. F. G. (1984) Eur. J. Biochem. 143, 227–234]. The remaining ten are new structures isolated from bronchial mucins; they are all extensions of the above‐mentioned nine oligosaccharides. These compounds are octasaccharide‐alditols containing the Y determinant together with the H determinant of either backbone‐type 1 or 2, and partial structures thereof:

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and 21b, which is 23c without Fucα(1→3), 18.2, which is 23c without any Fuc in the 6‐branch, and 22b, which is 23c without Fuc in the 3‐branch.
The development of rat α2‐macroglobulin
Authors: FLETCHER Sue , THOMAS Tim , SCHREIBER Gerhard , HEINRICH Peter C. , YEOH George C. T.
Published: March 2005

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute‐phase proteins. The levels of some acute‐phase proteins have been reported to increase in pregnant and tumour‐bearing animals.

Rat α2‐macroglobulin is classified as an acute‐phase protein. In this study we report the expression of α2‐macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, viceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of α2‐macroglobulin expression are localized by in situ hybridization of cDNA for α2‐macroglobulin to mid‐sagittal cryosections of rat embryos.

The level of mRNA coding for α2‐macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. α2‐Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. α2‐Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the α2‐macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting α2‐macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.