bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence study of the macrolide pentaene antibiotic filipin in aqueous solution and in a model system of membranes
Authors: CASTANHO Miguel A. R. B. , PRIETO Manuel J. E.
Published: March 2005

The polyene antibiotic filipin (a pentaene) has been studied using photophysical techniques. The polyene self‐aggregates in water with a critical micellar concentration of 2 μM. Two approaches were used to evaluate the aggregate dimensions: (a) a lower limit of 10 nm for the aggregate radius was obtained from energy transfer experiments; (b) a formula for rationalizing the turbidity spectrum was derived, and from its application a spherical shape of radius about 50 nm was deduced.

The low value for the fluorescence anisotropy of the aggregate (r= 0.02) is compatible with a very loose structure, i.e. the chromophore has very efficient depolarization dynamics that is not controlled by the aggregate size.

The Stern‐Volmer plot of aggregated filipin fluorescence quenching by iodide is non‐linear, presenting a downward curvature. A model was used for the interpretation of these data, along with a study of the quenching in transient state; it was concluded that all the components of the decay are affected by the quencher, i.e. the aggregate has a very open structure with respect to the iodide ion.

The partition constants of the polyene, Kp, between a model system of membranes (small unilamellar vesicles of dipalmitoylglycerophosphocholine) and the aqueous phase were determined from anisotropy measurements; the values obtained were Kp (gel phase) = (3.4 ± 0.8) × 103 and Kp (liquid crystal phase) = (7.7 ± 2.2) × 102. The observation that the polyene incorporation is efficient is at variance with the belief that the presence of sterols are essential for the interaction of polyene antibiotics with membranes [for review see Bolard, J. (1986) Biochim. Biophys. Acta 864, 257–304].
An improved procedure for reconstitution of the uncoupling protein and in‐depth analysis of H+/OH− transport
Authors: WINKLER Edith , KLINGENBERG Martin
Published: March 2005

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H+ uptake in K+‐deriven exchange diffusion quantitavely analyzed. UCP is isolated and reconstituted with medium‐length linear‐chain alkyl polyoxyethylene. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 μl/mg phospholipid with a vesicle diameter of 100 nm. One vescle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H+‐transport activity by UCP. Asolectin completely inactivates UCP. K+‐gradient‐driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H+ transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane‐bound H+ pools. Total H+ uptake after addition of carbonylcyanine m‐chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H+‐transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K+ electrode and 86Rb measurements. Recording Ψ using 3,3′‐diispropylthiadicarbocyanine shows a rapid generation of Ψ on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin. The Ψ collapses only after addition of external K+. By demonstrating that valinomycin‐induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first‐order rate, VH+/CH+, in which the rate, VH+, is related to H+‐uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on Ψ of H+ transport is measured by varying external K+ concentration. A virtually linear relation of the rate to the K+‐diffusion potential is observed, although the capacity is only slightly changed. The linear VH+/Ψ relationship resembles an open‐channel type of transport, but is discussed in terms of a low‐activation‐barrier type of carrier mechanism, in contrast to the log (VH+/Ψ) relation found for the ADP/ATP carrier with high activation barriers.
Polymxin B inhibits insulin‐induced glucose transporter and IGF II receptor translocation in isolated adipocytes
Authors: CORMONT Mireille , GREMEAUX Thierry , TANTI Jean‐François , VAN OBBERGHEN Emmanuel , LE MARCHAND‐BRUSTEL Yannick
Published: March 2005

In isolated adipocytes, polymyxin B inhibited insulin‐induced glucose incorporation into lipids in a dose‐dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin‐induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low‐density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin‐induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes.

Since low‐molecular‐mass GTP‐binding proteins have been implicated in the regulation of vesicular trafficking, we have used [α‐32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP‐binding proteins were revealed in plasma membrane and low‐density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low‐molecular‐mass GTP‐binding proteins in plasma membranes, but not in lowdensity microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor‐containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.
Enhanced cell‐free transcription of the ribosomal protein L32 gene by the polyoma virus enhancer PEA3 DNA‐binding protein
Authors: YOGANATHAN Thillainathan , COWIE Alison , HASSELL John A. , SELLS Bruce H.
Published: March 2005

The mouse‐ribosomal‐protein‐L32‐gene promoter contains a 12‐bp sequence motif within the 5′‐upstream region termed the β element which shows significant similarity with the consensus sequence of the polyoma‐virus‐enhancer PEA3. A cloned PEA3 DNA‐binding protein, expressed in Escherichia coli and purified, activates the expression of the ribosomal‐protein‐L32 gene in a cell‐free system. Moreover, the PEA3 protein participates in the formation of the ribosomal‐protein‐L32‐promoter – preinitiation‐transcription complex. The preinitiation complex formed with PEA3 is resistant to competition by oligonucleotides containing the β element. In addition anti‐PEA3 serum interacts with a factor in mouse L1210 nuclear extract that binds to the β element, causing a supershift in a mobility‐shift assay. Our study demonstrates for the first time that the PEA3 protein can transactivate a cellular gene in a cell‐free transcription system.
Spectral characteristics of cadmium‐containing phytochelatin complexes isolated from Schizosaccharomyces pombe
Authors: PLOCKE Donald J. , KÄGI Jeremias H. R.
Published: March 2005

Phytochelatins, heavy‐metal‐containing peptides with structures (γEC)nG, where n= 2–8, have been isolated from higher plants and the fission yeast Schizosaccharomyces pombe. The present work describes the isolation and characterization of several naturally occurring mixed complexes of these peptides from S. pombe exposed to 1 mM CdCl2. A lower‐molecular‐mass fraction from Sephadex G‐50 chromatography yielded three distinct species on further fractionation. HPLC chromatography revealed the presence of peptides with n= 1–4 in varying amounts in these three complexes, referred to as complexes I, II and III. Stoichiometries are proposed for these complexes, based on [Cd], [SH], [S2‐] and the amino acid content. Ultraviolet absorption and magnetic circular dichroism spectra of complexes II and III are similar, whereas the CD spectra of these two complexes are strikingly different. Compared to both complexes II and III, the CD bands of complex I are relatively weak. Ultraviolet absorption, CD and magnetic circular dichroism spectra provide a basis for the discussion of structural differences in these complexes.
The kidney as a novel target tissue for protein adduct formation associated with metabolism of halothane and the candidate chlorofluorocarbon replacement 2,2‐dichloro‐1,1,1‐trifluoroethane
Authors: HUWYLER Jörg , AESCHLIMANN Daniel , CHRISTEN Urs , GUT Josef
Published: March 2005

Hydrochlorofluorocarbons (HCFCs) have been identified as chemical replacements of the widely used chlorofluorocarbons (CFCs) that are implicated in stratospheric ozone depletion. Many HCFCs are structural analogues of the anesthetic agent halothane and may follow a common pathway of biotransformation and formation of adducts to protein‐centered and other cellular nucleophiles. Exposure of rats to a single dose of halothane (2‐bromo‐2‐chloro‐1,1,1‐trifluoroethane) or of the candidate CFC substitute HCFC 123 (2,2‐dichloro‐1,1,1‐trifluoroethane) led to the formation of trifluoroacetylated protein adducts (CF3CO‐proteins) not only in the liver, but also in the kidney as a novel target tissue for protein trifluoroacetylation. CF3CO‐proteins in the kidney amounted to about 5% of those formed in the liver of the same animal. The amount of CF3CO‐proteins formed within the kidney was roughly reflected by the capacity of metabolism of halothane or HCFC 123 by rat kidney microsomes in vitro which amounted to about 10% of that observed with liver microsomes. By immunohistochemistry, CF3CO‐proteins in the kidney were mainly localized in the tubular segments of the cortex. In the liver, the density of CF3CO‐proteins decreased from the central vein towards the portal triad. In vitro incubation of rat liver microsomes with halothane or HCFC 123 resulted in extensive formation of CF3CO‐proteins and reproduced faithfully the pattern of liver CF3CO‐proteins obtained in vivo. CF3CO‐proteins generated in vitro were immunochemically not discernible from those generated in vivo. Glutathione (5 mM) and cysteine (5 mM) virtually abolished CF3CO‐protein formation; the release of Br− from halothane and Cl− from HCFC 123 was reduced to much lesser a degree. S‐Methyl‐glutathione, N‐acetyl‐cysteine, methionine, and N‐acetyl‐methionine only slightly affected the formation of CF3CO‐proteins or metabolism of either substrate. The data suggest that metabolism and concomitant CF3CO‐protein formation of halothane or of candidate CFC replacements like HCFC 123 is not restricted to the liver but also takes place in the kidney. Furthermore, an in vitro system for CF3CO‐protein formation has been developed and used to show that protein‐centered and glutathione‐centered nucleophilic sites compete for intermediates of metabolism of halothane or of HCFC 123.
Structural requirements for the occupancy of pituitary adenylate‐cyclase‐activating‐peptide (PACAP) receptors and adenylate cyclase activation in human neuroblastoma NB‐OK‐1 cell membranes
Authors: ROBBERECHT Patrick , GOURLET Philippe , DE NEEF Philippe , WOUSSEN‐COLLE Marie‐Claire , VANDERMEERS‐PIRET Marie‐Claire , VANDERMEERS André , CHRISTOPHE Jean
Published: March 2005

In these structure activity studies, the 46 analogs of the 27‐amino‐acid form of the pituitary‐adenylate‐cyclase‐activating peptide, PACAP(1–27), and the 38‐amino‐acid form, PACAP(1–38), were either monosubstituted or bisubstituted at positions 1–3, 20 and 21 or N‐terminally shortened. All analogs were compared on human neuroblastoma NB‐OK‐1 cell membranes for their ability to occupy 125I‐[AcHis1]PACAP(1–27)‐labelled receptors (AcHis, Nα‐acetylhistidine) and to activate adenylate cyclase (in terms of potency and intrinsic activity). The monophasic slope of dose/effect curves on both parameters suggested interaction with one class of PACAP receptor. Residues 28–38 in the C‐terminally extended peptide, PACAP(1–38), played a favorable role in recognition, in that receptors coupled to adenylate cyclase were, in general, more sensitive to PACAP(1–38) analogs than to the corresponding PACAP(1–27) analogs. At variance with PACAP(6–27), PACAP(6–38) was well recognized and acted as a potent competitive antagonist (Ki 1.5 nM).

Residues 1–3 were all important in enzyme activation: modification of the β‐turn potential gave full agonists (the LAla2 and DAla2 derivatives) or partial agonists (LPhe2 and DPhe2; LArg2 and DArg2; Glu3 and Asn3). Finally, a proper α‐helix was also important: the combined substitution of Lys21/Lys22 by Gly21/Gly22 decreased the binding affinity sharply.
Molecular cloning, stage‐specific expression and cellular distribution of a putative protein kinase from Plasmodium falciparum
Authors: ZHAO Yi , KAPPES Barbara , YANG Jiewu , FRANKLIN Richard M.
Published: March 2005

A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A + T)‐rich region and transcribed in a different orientation. No intron sequence exists in PfPK2. The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases. Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium‐calmodulin‐dependent protein kinase, human protein kinase C, and bovine cAMP‐dependent protein kinase, respectively. Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits. Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage‐specific fashion and mainly localized in the parasitic membrane.
Evidence for a sialosyl cation transition‐state complex in the reaction of sialidase from influenza virus
Authors: CHONG Andrew K. J. , PEGG Michael S. , TAYLOR Neil R. , von ITZSTEIN Mark
Published: March 2005

The enzyme mechanism of sialidase from influenza virus has been investigated by kinetic isotope methods, NMR, and a molecular dynamics simulation of the enzyme‐substrate complex. Comparison of the reaction rates obtained with the synthetic substrate 4‐methylumbelliferyl‐N‐acetyl‐α‐D‐neuraminic acid and the [3,3‐2H]‐substituted substrate revealed β‐deuterium isotope effects for V/Km ranging over 1.09–1.15 in the pH range 6.0–9.5, whereas the effects observed for V in this pH range increased from 0.979 to 1.07. In D2O, βDV/Km was slightly increased by 2% and 5% at pD 6.0 and 9.5 respectively, while βDV was unchanged. Solvent isotope effects of 1.74 were obtained for both βDV/Km and βDV at pD 9.5, with βDV/Km decreasing and βDV remaining constant at acidic pD. 1H‐NMR experiments confirmed that the initial product of the reaction is the α‐anomer of N‐acetyl‐D‐neuraminic acid. Molecular dynamics studies identified a water molecule in the crystal structure of the sialidase‐N‐acetyl‐D‐neuraminic acid complex which is hydrogen‐bonded to Asp151 and is available to act as a proton donor source in the enzyme reaction. The results of this study lead us to propose a mechanism for the solvent‐mediated hydrolysis of substrate by sialidase that requires the formation of an endocyclic sialosyl cation transition‐state intermediate.
Functional analysis of primers and templates in the synthesis of DNA catalyzed by human immunodeficiency virus type 1 reverse transcriptase
Authors: NEVINSKY Georgyi A. , ANDREOLA Marie‐Line , JAMKOVOY Vitalyi I. , LEVINA Asja S. , BARR Philip J. , TARRAGO‐LITVAK Laura , THARAUD Danièle , LITVAK Simon
Published: March 2005

The kinetics of copying of poly(A) · (dT)n, poly(A) · (U)n, poly(dA) · (dT)n and poly(A) · (dT)9‐U by reverse transcriptase of human immunodeficiency virus‐1 (HIV‐1) has been studied and the binding affinity of the enzyme, for template or primer, determined. Short oligonucleotides and dTTP served as primers in the HIV‐1 reverse‐transcriptase‐dependent DNA synthesis. Km and Vmax were measured as functions of the primer chain length; the logarithm of the values of both Km and Vmax increased linearly up to 10. For longer primers (n= 11 to n= 24) the increase of those values changes very little. The enhanced affinity of the primers, (dT)n or (U)n due to the formation of one complementary pair, A · dT, dA · dT, A · U was estimated as a factor of 2. A specific property of HIV‐1 reverse transcriptase compared with other DNA polymerases (procaryotes, eucaryotes, other retroviruses and archaebacteria) was its higher affinity to riboprimers as compared to deoxyriboprimers.

Relative initial rates when copying poly(A) or poly(dA) templates using different primers and various conditions were compared; the optimal temperature for the reaction of polymerization with poly(A) or poly(dA) templates and (U)10, (dT)10 or (dT)9‐U primers was determined. The maximal activity of the enzyme in the case of poly(A) and decanucleotide primers was found at temperatures between 27–31°C. An increase in the primer length results in the stabilization of the template · primer duplex complexed to the enzyme, thus increasing to more than 40°C the optimal temperature of polymerization. The activation energy (Ea) values of the polymerization reaction for different template · primer complexes were evaluated.
Molecular and metabolic changes in white adipose tissue of the rat during development of ventromedial hypothalamic obesity
Authors: COUSIN Béatrice , AGOU Karen , LETURQUE Armelle , FERRE Pascal , GIRARD Jean , PÉNICAUD Luc
Published: March 2005

We have previously shown that rats made obese by lesion of ventromedial hypothalamus (VMH) nuclei, demonstrate an hyper‐responsiveness to insulin with regard to whole‐body glucose utilization one week after injury. This is mainly due to an increased glucose uptake in white adipose tissue. Six weeks after the lesion, glucose utilization in white adipose tissue returns to normal values. These modifications in insulin responsiveness could be mediated by altered activity and/or concentration of intracellular insulin effectors. In this study, we have measured the expression of the insulin‐sensitive glucose transporter, Glut 4 and the activities and expression of key lipogenic enzymes (fatty‐acid synthase and acetyl‐CoA carboxylase) in white adipose tissue, one and six weeks after the lesion. All these parameters, as well as glucose transport and metabolism determined in white adipocytes, were markedly increased one week after the lesion. They returned to control values within six weeks in VMH‐lesioned rats. These results indicate the existence of an increased expression of Glut 4 and lipogenic enzymes in white adipose tissue of VMH‐lesioned rats which decreased with time and were parallel to glucose utilization determined in vivo.
Phorbol esters, bombesin and insulin elicit differential responses on the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase system in primary cultures of foetal and adult rat hepatocytes
Authors: CASCALES Maria , MARTIN‐SANZ Paloma , BOSCA Lisardo
Published: March 2005

The effects of 4β‐phorbol 12‐myristate 13‐acetate (PMA), bombesin and insulin on 6‐phosphofructo‐2‐kinase (PFK‐2) activity, on fructose 2,6‐bisphosphate concentration and on the phosphorylation state of PFK‐2 were investigated in primary cultures of hepatocytes from foetal and adult rats. Bombesin stimulated PFK‐2 activity and increased hexose phosphate (glucose 6‐phosphate and fructose 6‐phosphate) and fructose 2,6‐bisphosphate content in hepatocytes both in the foetal and adult state. However, PMA‐treated foetal cells exhibited a marked stimulation in fructose 2,6‐bisphosphate concentration and in PFK‐2 activity as well as in the content of hexose phosphates, while no response was found in the case of adult hepatocytes. Moreover, the effect of PMA on foetal hepatocytes was suppressed when cells were incubated with cycloheximide, but not when this effect was elicited by bombesin or insulin. These results, and those obtained on the phosphorylation state of PFK‐2, suggest that there are different pathways that modulate fructose 2,6‐bisphosphate content and, therefore, the control mechanisms of glycolysis and gluconeogenesis at this regulatory step, both in adult and foetal rat liver.
Molecular cloning of a glucoamylase gene from a thermophilic Clostridium and kinetics of the cloned enzyme
Authors: OHNISHI Hiroshi , KITAMURA Hiroki , MINOWA Takashi , SAKAI Hiroshi , OHTA Takahisa
Published: March 2005

Clostridium sp. G0005 produces a cell‐bound glucoamylase (CGA).The gene encoding CGA has been sequenced. The deduced amino acid sequence begins with a putative 21‐residue signal sequence for secretion of bacterial lipoproteins, which suggests that a putative CGA precursor is modified and secreted like other bacterial lipoproteins in Clostridium sp. G0005, and that the modified residue is important in the cell‐bound form of mature CGA. Comparison of the amino acid sequence of the CGA precursor with known eukaryotic enzymes showed several regions of high similarity in spite of low similarity throughout the primary structure. CGA is the first bacterail glucoamylase to be cloned.

The CGA gene was expressed in Escherichia coli with an inducible expression plasmid, in which the 5′ non‐coding region and the N‐terminal coding region of the gene were replaced with the lac promoter. Kinectic studies of the cloned enzyme purified from E.coli were performed with a set parameters. CGA had typical kinectic properties for a glucoamylase, but this bacterial enzyme had higher isomaltose‐hydrolyzing activity than other eukaryotic glucoamulases.
Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non‐stoichiometric oxidation of NADPH in the presence of oxygen
Authors: KUMAR Sushil , BJÖRNSTEDT Mikael , HOLMGREN Arne
Published: March 2005

The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox‐active dithiols. We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 μM selenite plus 0.05 μM calf thymus thioredoxin reductase at pH 7.5 caused a non‐stoichiometric oxidation of NADPH (100 μM after 30 min). In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 μM E. coli thioredoxin also resulted in non‐stoichiometric oxidation of NADPH, consistent with oxidation of the two active‐site thiol groups in thioredoxin to a disulfide. Kinetically, the reaction was complex with a lag phase at low selenite concentrations. Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s). Ferricytochrome c was effectively reduced by calf thymus thioredoxine reductase and selenite in the presence of oxygen. Selenite caused a strong dose‐dependent inhibition of the formation of thiol groups from insulin disulfides with either the E. coli or calf‐thymus thioredoxin system. Thus, under aerobic conditions selenite catalyzed, NADPH‐dependent redox cycling with oxygen, a large oxygen‐dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide‐reductase activity.
Dependence of the adenovirus tripartite leader on the p220 submit of eukaryotic initiation factor 4F during in vitro translation
Authors: THOMAS Adri A. M. , SCHEPER Gert C. , KELEIJN Miranda , DE BOER Mariska , VOORMA Harry O.
Published: March 2005

The adenovirus tripartite leader (TPT) 5′ untranslated region (5′UTR) allows translation in poliovirus‐infected cells, in which the p220 subunit of eukaryotic initiation factor 4F is degraded. This p220‐independent translation was investigated by measuring in vitro translation in a reticulocyte lysate of a report gene, chloramphenicol acetyltransferase, coupled to the TPT 5′UTR. The p220 subunit was degraded by translation of a foot‐and‐mouth‐disease L‐protease construct. Surprisingly, the TPT 5′UTR was dependent on intact p220, as are other naturally capped mRNA species. Translation of encephalomyocarditis virus RNA was p220 independent, as expected from its ability to support internal, cap‐independent initiation. In vitro protein‐synthesis experiments with purified initiation factors confirmed the dependence of TPT mRNA translation on eukaryotic initiation factor 4F. The relationship between adenovirus TPT‐5′UTR‐directed translation and poliovirus‐induced host cell shut‐off is discussed.
Escherichia coli Rep protein and helicase IV
Authors: YANCEY‐WRONA Janet E. , WOOD Edger R. , GEORGE James W. , SMITH Karen R. , MATSON Steven W.
Published: March 2005

Rep protein and helicase IV, two DNA‐dependent adenosine 5′‐triphosphatases with helicase activity, have been purified from Escherichia coli and characterized. Both enzymes exhibit a distributive interaction with single‐stranded DNA as DNA‐dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition of E. Coli single‐stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119‐bp or 343‐bp duplex region was substantially reduced compared to unwinding of the 71‐bp substrate. At each concentration of protein examined, the number of base paris unwound was greatest using the 71‐bp substrate, intermediate with the 119‐bp substrate and lowest using the 343‐bp substrate. The addition of E. coli SSB did not increase the fraction of the 343‐nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2 or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that, in vitro, and in the absence of addition proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.
S100P, a novel Ca2+ ‐binding protein from human placenta
Authors: BECKER Tilmann , GERKE Volker , KUBE Eckhard , WEBER Klaus
Published: March 2005

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca2+‐binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identify with the brain S100 proteins α and β, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/plypeptide chain. The low‐affinity site (Kd= 800 μM), which, in analogy to S100β, seems to involve the N terminal EF hand, can be followed by the Ca2+ ‐dependent decrease in tyrosine fluorescence. The high‐affinity site., provided by the C‐terminal EF hand, influences the reactivity of the sole cysteine which is located in the C‐terminal extension (Cys85). Binding to the high‐affinity site (Kd= 1.6 μM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6‐proprionly‐2‐dimethylaminonaphtalene (Prodan). The Prodan fluorescence shows a Ca2+ ‐dependent red shift of the maximum emisson wavelength from 485 nm to 502 nm, which is accomplished by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvant in Ca2+ ‐bound S100P, making this region of the molecule, the so‐called C‐terminal extention with a cellular target. In p11, a different member of the S100 family, C‐terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca2+ ‐independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca2+ ‐dependent conformational change which involves hydrophobic residues of the C‐terminal extension.
Type XIV collagen is a varient of undulin
Authors: TRUEB Judith , TRUEB Beat
Published: March 2005

We have isolated undulin, an extracellar matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecularmolecular mass of about 600 kDa and is composed of three 210‐kDa subunits linked by reducible as well as non‐redulic bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple‐helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity‐purified antibodies yieded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones terminus both polypeptides contained several similar repeats related to the III modules found in fibronectin. Towards the carboxyl terminus, however the two sequences diverged substantially from each other. While the human sequence termineted in a proline‐rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 fo type IX collagen and with a typical collagenous triple helix, A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple‐helical domain, as found in type XIV collagen.
The role of retinal in the thermal stability of the purple membrane
Authors: CLADERA Josep , GALISTEO M. Luisa , SABÉS Manuel , MATEO Perdro L. , PADRÓS Esteve
Published: March 2005

Differential scanning calorimetry demonstrates that the bleached form of the purple membrane does not possess any measurable thermal transition in water, up to 105°C, Whereas in 0.1 M phosphate pH 7.5 it shows at about 82°C, with an enthalpy of 110 KJ/mol. In the latter medium, the native membrane shows the main transition at 97°C with an enthalpy of 390kJ/mol. The reduced form of the purple membrane shows two small transitions in water, as well as in 0.1 M Phosphate, which do not seem to be related to the main thermal transition of the native membrane. Fouriertransform infrared spectra in D2O show that the two modified samples, as well as the native one, undergo similar secondary structural changes upon thermal denaturation. these changes appear to extend through a wide temperature range for both modified forms, particularly for the bleached one. The results suggest that the main thermal transition in the purple membrane is due to a cooperative conformational change involving the disruption of the network of electrostatic and hydrogen‐bonding interactions was originate from the protonated Schiff base. In the two modified membranes, these conformational changes appear to proceed smoothly through a rather low or non‐cooperative process. The thermal behaviour of the bleached membrane in water resembles that of the molten glubule state described for several globular protiens.
A rat cerebellar protein containing the cdc10/SWI6 motif
Authors: TAOKA Masato , YAMAKUNI Tohru , SONG Si‐Young , YAMAKAWA Yoshio , SETA Kazuo , OKUYAMA Tsuneo , ISOBE Toshiaki
Published: March 2005

Systematic analysis of soluble proteins in developing rat cerebellum by an automated two‐dimensional liquid‐chromatography system detected a number of proteins which increased transiently during the initial stage of postnatal development. One of the proteins, V‐1 was isolated using a liquid‐chromatography system, and its amino acid sequence was determined by analysis of the purified protein. The sequence showed that the V‐1 protein consisted of 117 amino acids with an acetylated N‐terminus, and has 2.5 internal sequence repeats of 33 amino acids. Computer retrieval of the sequence indicated that the repeated sequences have a structural characteristics of the cdc10/SWI6 motif, which is found in a series of proteins, including those involved in cell‐cycle control and cell‐fate determination in yeast, Drosophila melanogaster and Caenorhabditis elegans. The structure of V‐1, coupled with its controlled expression in early postnatal development, implies a potential role for V‐1 in cerebellar morphogenesis.
Prosomes and their multicatalytic proteinase activity
Authors: NOTHWANG Hans‐Gred , COUX Oliver , BEY Fayçal , SCHERRER Klaus
Published: March 2005

Prosomes were first described as being mRNA‐associated RNP (ribonucleoprotein) particles and subcomponents of repressed mRNPs (messenger ribonucleoprotein). We show here that prosomes isolated from translationally inactive mRNP have a protease activity identical to that described by others for the multicatalytic proteinase complex (MCP, ‘proteasome’). By RNase or non‐ionic detergent treatment, the MCP activity associated with repressed non‐globin mRNP from avian erythroblasts, sedimenting at 35 S, could be quantitatively shifted on sucrose gradients to the 19‐S sedimentation zone characteristic of prosomes, which were identified by monoclonal antibodies. The presence of small RNA in the enzymatic complex was shown by immunoprecipitation of the protease activity out of dissociated mRNP using a mixture of anti‐prosome monoclonal antibodies; a set of small RNAs 80–120 nucleotides long was isolated from the immunoprecipitate. Furthermore, on CsCl gradients, colocalisation of the MCP activity with prosomal proteins and prosomal RNA was found, and no difference in the prosomal RNA pattern was observed whether the particles were fixed or not prior to centrifugation. These data indicate that the MCP activity is a property of prosomes, shown to be in part RNP and subcomplexes of in vivo untranslated mRNP. A hypothesis for the role of the prosome‐MCP particles in maintaining homeostasis of specific protein levels is proposed.
Determination of DNA‐binding parameters for the Bacillus subtilis histone‐like HBsu protein through introduction of fluorophores by site‐directed mutagenesis of a synthetic gene
Authors: GROCH Nicolas , SCHINDELIN Hermann , SCHOLTZ Andreas S. , HAHN Ulrich , HEINEMANN Udo
Published: March 2005

A synthetic gene encoding the histone‐like DNA‐binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild‐type protein. N‐terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli.. Site‐directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA‐binding parameters by fluorescence spectroscopy. According to gel‐retardation experiments, the mutant protein [Phe47 → Trp]HBsu shows identical DNA binding to wild‐type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47 → Trp]HBsu binds double‐stranded DNA with a dissociation constant in the micromolar range. Computer‐assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47 → Trp]HBsu appears to contact three or four base pairs of DNA. These results are in pritial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.
Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase
Authors: WHITE Malcolm F. , FOTHERGILL‐GILMORE Linda A.
Published: March 2005

A system has been developed to allow the convenient production, expression and purification of site‐directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild‐type activity. A simple one‐column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active‐site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 * 104) in the catalytic efficiency, and an 11‐fold increase in the Km for the cofactor 2,3‐bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.
Evidnece that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in‐phase AUG codons
Authors: SUZUKI Tamino , YOSHIDA Tadashi , TUBOI Syozo
Published: March 2005

Rat liver contains two isozymes of fumarase, mitochondrial and cytosolic enzymes. Recently, we suggested that the precursors of both isozymes might be synthesized by one species of mRNA [Suzuki. T., Sato, M., Yoshida, T. & Tuboi, S. (1989) J. Biol. Chem. 264, 2581–2586]. To examine this possibility, we have isolated and characterized rat genomic clones for fumarase. The isolated clones covered almost all of the 5′ half of the fumarase gene consisting of five exons. The first exon contained the whole 5′ non‐coding region and the signal peptide of mitochondrial precursor. The second exon encoded 45 amino acid residues of both mature proteins, starting from the N‐terminal alanine. By using the boundary region of the first intron and the second exon as an S1‐nuclease‐analysis probe, we obtained conclusive evidence that rat liver contains no other mRNA specific for the cytosolic isozyme of fumarase. Two transcription‐initiation sites were identified by further S1‐nuclease‐mapping analysis and were shown to be located very close to each other, differing by only four bases in length. Therefore, these sites were considered to be functionally the same. The results obtained by hybrid‐selected translation, with a DNA fragment of the 5′ non‐coding region as a hybridization probe for selecting mRNA, were consistnet with the above findings. We found a plausible secondary structure within the 5′ non‐coding mRNA sequence that may impede initiation and so alter the efficiency of translation. We also discuss the mechanism regulating translational initiation.
The primary structure of the α subunit of a starfish guanosine‐nucleotide‐binding regulatory protein involved in 1‐methyladenine‐induced oocyte maturation
Authors: CHIBA Kazuyoshi , TADENUMA Hirohiko , MATSUMOTO Midori , TAKAHASHI Katsunobu , KATADA Toshiaki , HOSHI Motonori
Published: March 2005

Starfish‐oocyte maturation induced by 1‐methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX‐catalyzed ADP‐ribosylation of a 39‐kDa guanosine‐nucleotide‐binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde‐induced signals operate via a membrane receptor and are carried by the PTX‐sensitive G protein. When PTX‐injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the α subunit of an inhibitory rat G protein (Gi‐2), A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat Gi‐2 cDNA. The deduced amino acid sequence was 85% and 89% identical to rat Gi‐2 and rat Gi‐1, respectively. The α subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the α subunit of the PTX‐sensitive G protein in oocytes. The C‐terminal sequence, KNNLKDCGLF, was identical to that of Gi, suggesting that the cysteine residue is the site of ADP‐ribosylation.
A major oligomeric fibroblast proteoglycan identified as a novel large form of type‐XII collagen
Authors: KOCH Manuel , BERNASCONI Carlo , CHIQUET Matthias
Published: March 2005

Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600‐kDa polypeptide is likely to be a dimer of two smaller subunits which are cross‐linked by non‐reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350‐kDa and 600‐kDa (but not the 300‐kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S]sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non‐collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type‐XII collagen from its full‐length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209–221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type‐XII collagen: its subunits are larger by one third, and it is a proteoglycan.
Inactivation of liver 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase by phenylglyoxal
Authors: RIDER Mark H. , HUE Louis
Published: March 2005

Treatment of liver 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase with the arginine‐specific reagent, phenylglyoxal, irreversibly inactivated both 6‐phosphofructo‐2‐kinase and fructose‐6‐bisphosphatase in a time‐dependent and dose‐dependent manner. Fructose 6‐phosphate protected against 2,6‐phosphofructo‐2‐kinase inactivation, whereas MgGTP protected against fructose‐2,6‐bisphosphatase inactivation. Semi‐logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non‐linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 mol/mol enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic‐AMP‐dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic‐AMP‐dependent protein kinase interacts with a modified arginine residue. Chymotryptic clcavage of the modified protein and microsequencing showed that Arg225, in the 6‐phosphofructo‐2‐kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6‐phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6‐phosphate binding in the 6‐phosphofructo‐2‐kinase domain of the bifunctional enzyme.
Characterisation by mass spectrometry and 1H‐NMR of novel hexasaccharides among the acidic O‐linked carbohydrate chains of bovine submaxillary mucin
Authors: CHAI Wengang , HOUNSELL Elizabeth F. , CASHMORE Geoffrey C. , ROSANKIEWICZ Jerzy R. , FEENEY James , LAWSON Alexander M
Published: March 2005

The acidic oligosaccharide alditols released from bovine submaxillary‐gland mucin by Carlson degradation were investigated by a combination of liquid secondary‐ion mass spectrometry, methylation analysis and 1H‐NMR. Among the largest structures identified were four branched hexasaccharides, three of them novel, comprising two separate pairs of structures. One pair contained the sequence Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–) (Fuc, l‐fucose), at C3 of N‐acetylgalactosaminitol and differed only by substitution at C6 by N‐acetylneuraminic or N‐glycolylneuraminic acid. The other pair also differed in substitution of the sialic acid linked at C6 and contained the GalNAc(α1–3)[Fuc(α1–2)]Gal(β1–4)GlcNAc(β1–), sequence at C3 of N‐acetylgalactosaminitol. The Lewisy and blood‐group‐A determinants of these sequences have not been found previously in the acidic oligosaccharides of bovine submaxillary‐gland mucin, although they have recently been characterised in the neutral chains of bovine submaxillary‐gland mucin.
Primary structure of neutral and acidic oligosaccharide‐alditols derived from the jelly coat of the Mexican axolotl
Authors: STRECKER Gérard , WIERUSZESKI Jean‐Michel , MICHALSKI Jean‐Claude , ALONSO Catherine , LEROY Yves , BOILLY Benoni , MONTREUIL Jean
Published: March 2005

Six major neutral and acidic oligosaccharide‐alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3‐deoxy‐d‐glycero‐d‐galacto‐nonulosonic acid (dNloA) and l‐fucose (Fuc). The structures of the six major oligosaccharides were established as follows:

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