bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and characterization of a novel thermostable 4‐α‐glucanotransferase of Thermotoga maritima cloned in Escherichia coli
Authors: LIEBL Wolfgang , FEIL Robert , GABELSBERGER Josef , KELLERMANN Josef , SCHLEIFER Karl‐Heinz 
Published: March 2005

Maltodextrin glycoslyltransferase (4‐α‐glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4‐α‐glucanotransferase, an approximately 53‐kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55°C up to 80°C.The proposed action modus is the transfer of 1,4‐α‐glucanosyl chains, thus resulting in the disproportionation of 1,4‐α‐glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low‐molecular‐mass malto‐oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto‐oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4‐α‐glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues.Comparison of the N‐terminal amino acid sequence of 4‐α‐glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N‐termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch‐ and maltodextrin‐converting enzymes.
Site‐directed mutagenesis of the Streptomyces R61 DD‐peptidase
Authors: HADONOU Ayaovi M. , WILKIN Jean‐Marc , VARETTO Louis , JORIS Bernard , LAMOTTE‐BRASSEUR Josette , KLEIN Daniel , DUEZ Colette , GHUYSEN Jean‐Marie , FRÈRE Jean‐Marie 
Published: March 2005

The importance of various residues in the Streptomyces R61 penicillin‐sensitive DD‐peptidase has been assessed by site‐directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65→Arg mutant with the peptide and thiolester substrates was decreased 100–200‐fold and the rate of penicillin inactivation was decreased 20 000‐fold or more. The mutant thus behaved as a poor, but penicillin‐resistant, DD‐peptidase.The other studied mutations, the mutations Phe58→Leu, Tyr90→Asn, Thr101→Asn, Phe164→Ala, Asp225→Glu and Asp225→Asn had little influence on the catalytic and penicillin‐binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164→Ala mutant was significantly more unstable than the wild‐type enzyme.
A comparison of the catalytic properties of cellobiose: quinone oxidoreductase and cellobiose oxidase from Phanerochaete chrysosporium
Authors: SAMEJIMA Masahiro , ERIKSSON Karl‐Erik L. 
Published: March 2005

Several catalytic properties of the FAD enzyme cellobiose:quinone oxidoreductase (CBQ) and the heme/FAD enzyme, cellobiose oxidase (CBO) have been investigated and compared. Dichlorophenolindophenol was found to be a very good electron acceptor for cellobiose oxidation by both enzymes. The optimal pH value for this oxidation with dichlorophenol‐indophenol as a co‐substrate was observed around pH 4 for both enzymes. The turnover numbers of this reaction were also very similar. The Km values for cellobiose oxidation were identical, whereas the Km for CBO with dichlorophenol‐indophenol is lower than that of CBQ.Atmospheric oxygen is a very poor electron acceptor for both CBO and CBQ, however, CBO can utilize cytochrome c as an effective electron acceptor, while CBQ cannot. The specific activity of CBO for cytochrome c is thus about 200‐times higher than for oxygen. Thus, one way to distinguish the two enzymes is by the cytochrome‐c‐reducing ability of CBO. Therefore, we propose that the nomenclature for CBO is tentatively changed to cellobiose:cytochrome c oxidoreductase until a rational name can be installed.Both enzymes have radical‐reducing activities. The cation radical, derived from 1,2,4,5‐tetramethoxybenzene, was reduced by both enzymes at almost the same reaction rate. The phenoxyradical produced by lignin peroxidase, catalyzing the oxidation of acetosyringon, was also reduced by both enzymes. The reduction of phenoxyradicals formed by phenoloxidases (lignin peroxidases, as well as laccases) may be important in preventing repolymerization reactions which we suggest would significantly facilitate lignin degradation.
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].
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.
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.
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 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.
Purification, phosphorylation and control of the guanine‐nucleotide‐exchange factor from rabbit reticulocyte lysates
Authors: OLDFIELD Susan , PROUD Christopher G. 
Published: March 2005

A simple, improved procedure for the isolation of guanine‐nucleotide‐exchange factor (GEF) and for eukaryotic initiation factor 2 (eIF‐2) from rabbit reticulocyte lysates has been developed using ion‐exchange chromatography on S‐Sepharose, Q‐Sepharose, Mono Q and Mono S. The majority of the eIF‐2 is separated from GEF at an early stage in the procedure and the remaining small amount of eIF‐2 · GEF complex is separated from the bulk of the GEF by FPLC on Mono S. The procedure yields approximately 2 mg each of eIF‐2 and GEF, of 90% and greater than 80% purity, respectively, from the blood of ten rabbits. All fractions of purified GEF contain four subunits of molecular masses 84, 66, 54 and 39 kDa, with various amounts of a fifth, 30‐kDa subunit.The modulation of GEF activity was investigated using the highly purified factor in a guanine‐nucleotide‐exchange assay. The activity of GEF was stimulated by physiological concentrations of the polyamines, spermine and spermidine, but was unaffected by another polycationic compound, polylysine. Activity was also found to be inhibited by 1 mM NADP+ or NAD+, and this inhibition was overcome by the presence of 1 mM NADPH. Stoichiometric amounts of GEF were unable to release GDP from eIF‐2 · GDP complexes in the absence of free guanine nucleotides, suggesting that GEF operates by a ternary‐complex mechanism.Casein kinase 1 or casein kinase 2 can each phosphorylate the largest subunit (84 kDa) of GEF. These enzymes both phosphorylate serine residues in GEF but they phosphorylate distinct sites, as demonstrated by phosphopeptide mapping following proteolytic or cyanogen bromide digestion. Neither of these kinases phosphorylated any of the other subunits of GEF to any significant extent and several other kinases were inactive against GEF. No effect of phosphorylation on activity could be demonstrated.
Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site‐directed mutagenesis and photolabelling experiments
Authors: HERVÉ Mireille , PILLET Laurence , HUMBERT Paul , TRÉMEAU Odile , DUCANCEL Frédéric , HIRTH Christian , MÉNEZ André 
Published: March 2005

The positive charge of Lys27 was supressed by chemical means in two short‐chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin α from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6–15‐fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site‐directed mutagenesis. This change provokes a 120‐fold decrease in the affinity, which reflects a major alteration of toxin‐receptor cognate events.Using toxin‐α derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor‐bound state by photocoupling experiments. The δ chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the α chain. The toxin derivative weakly labelled the α and γ chains but not the β chain. The toxin may therefore interact with subunits other than the α chain, at least in the vicinity of Lys27.
The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C
Authors: BAKKER Harry M. , TANS Guido , JANSSEN‐CLAESSEN Truus , THOMASSEN M. Christella L. G. D. , HEMKER H. Coenraad , GRIFFIN John H. , ROSING Jan 
Published: March 2005

Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC‐catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first‐order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second‐order rate constant of 6.1 × 105 M−1 s−1. Stimulation of APC‐catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230‐fold) of factor Va inactivation was observed with 10 μM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC‐catalyzed factor Va inactivation was observed.Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine ≤ oleic acid ≤ phosphatidic acid ≤ phosphatidylglycerol ≤ phosphatidylmethanol ≤ phosphatidylserine. APC‐catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis‐Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 μM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 μM phospholipid. The Vmax increased from 20 mol factor Va inactivated · min−1· mol APC−1 at 2.5 μM phospholipid to 62 mol factor Va inactivated · min−1· mol APC−1 at 10 μM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC‐catalyzed factor Va inactivation. Protein‐S‐dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC‐catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.
A reinvestigation of the pre‐steady‐state ATPase activity of the nitrogenase from Azotobacter vinelandii
Authors: MENSINK Richard E. , WASSINK Hans , HAAKER Huub 
Published: March 2005

The pre‐steady‐state ATPase activity of nitrogenase has been reinvestigated. The exceptionally high burst in the hydrolysis of MgATP by the nitrogenase from Azotobacter vinelandii communicated by Cordewener et al. (1987) [Cordewener J., ten Asbroek A., Wassink H., Eady R. R., Haaker H. & Veeger C. (1987) Eur. J. Biochem. 162, 265–270] was found to be caused by an apparatus artefact. A second possible artefact in the determination of the stoichiometry of the pre‐steady‐state ATPase activity of nitrogenase was observed. Acid‐quenched mixtures of dithionite‐reduced MoFe or Fe protein of Azotobacter vinelandii nitrogenase and MgATP contained phosphate above the background level. It is proposed that due to this reaction, quenched reaction mixtures of nitrogenase and MgATP may contain phosphate in addition to the phosphate released by the ATPase activity of the nitrogenase complex.It was feasible to monitor MgATP‐dependent pre‐steady‐state proton production by the absorbance change at 572 nm of the pH indicator o‐cresolsulfonaphthalein in a weakly buffered solution At 5.6°C, a pre‐steady‐state phase of H+ production was observed, with a first‐order rate constant of 2.2 s−1, whereas electron transfer occurred with a first‐order rate constant of 4.9 s−1. At 20.0°C, MgATP‐dependent H+ production and electron transfer in the pre‐steady‐state phase were characterized by observed rate constants of 9.4 s−1 and 104 s−1, respectively. The stopped‐flow technique failed to detect a burst in the release of protons by the dye‐oxidized nitrogenase complex.It is concluded that the hydrolysis rate of MgATP, as judged by proton release, is lower than the rate of electron transfer from the Fe protein to the MoFe protein.