bluuebunny/crossref_metadata_2025 huggingface dataset:
Import and processing of heart mitochondrial cyclophilin D
Authors: Johnson Nicholas , Khan Ayesha , Virji Sukaina , Ward John M. , Crompton Martin
Published: March 2003

Cyclophilins are a family of cyclosporin‐A‐binding proteins which catalyse rotation about prolyl peptide bonds. A mitochondrial isoform in mammalian cells, cyclophilin D, is a component of the permeability transition pore that is formed by the adenine nucleotide translocase and the voltage‐dependent anion channel at contact sites between the inner and outer membrane. This study investigated the submitochondrial location of cyclophilin D by following the fate of radiolabelled protein following import. Precursor [35S]cyclophilin D was expressed in vitro from a PCR‐generated cDNA. The precursor was imported by rat heart mitochondria and processed in a single step to a 21‐kDa protein that was identical (SDS/PAGE) to an in vitro expressed mature protein and a cyclophilin D purified from rat heart mitochondria. No further modification of the mature protein could be demonstrated. Fractionation of mitochondria following import established that cyclophilin D locates only to the matrix. It is concluded that cyclophilin D binding to the permeability transition pore must occur at the inner face of the mitochondrial inner membrane.
Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis‐deficient yeast cells does not require the Drs2 protein
Authors: Marx Uwe , Polakowski Thomas , Pomorski Thomas , Lang Christine , Nelson Hannah , Nelson Nathan , Herrmann Andreas
Published: March 2003

Evidence is presented that endocytosis‐deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1‐palmitoyl‐2‐{6‐[7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl(NBD)amino] caproyl}phosphatidylcholine (P‐C6‐NBD‐PtdCho) and P‐C6‐NBD‐phosphatidylserine (P‐C6‐NBD‐PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half‐time of < 15 min at 0 °C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein‐mediated, as the flip‐flop of both analogues was very slow in liposomes composed of plasma‐membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P‐C6‐NBD‐PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P‐C6‐NBD‐PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4Δdrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P‐C6‐NBD‐PtdCho and P‐C6‐NBD‐PtdSer, respectively, became internalized within 15 min at 0 °C. The preferential orientation of P‐C6‐NBD‐PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild‐type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.
Phosphorylation, dephosphorylation and DNA‐binding of the Bradyrhizobium japonicum RegSR two‐component regulatory proteins
Authors: Emmerich Ralf , Panglungtshang Kelnor , Strehler Philipp , Hennecke Hauke , Fischer Hans‐Martin
Published: March 2003

Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox‐responsive transcriptional activator NifA which is encoded in the fixR–nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position −68 in the fixR–nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity‐purified RegR and a truncated soluble variant of RegS (RegSC), both as N‐terminally His6‐tagged proteins. RegSC autophosphorylated when incubated with [γ‐32P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegSC exhibited phosphatase activity on RegR‐phosphate. Chemical stability tests and site‐specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N‐terminal but not the C‐terminal domain of RegR interacts with RegSC. Band‐shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA‐binding activity compared with dephosphorylated RegR or the mutant protein RegR‐D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two‐component regulatory systems.
Effect of glucose and deoxyglucose on the redistribution of calcium in Ehrlich ascites tumour and Zajdela hepatoma cells and its consequences for mitochondrial energetics
Authors: Wojtczak Lech , Teplova Vera V. , Bogucka Krystyna , Czyż Aneta , Makowska Agnieszka , Więckowski Mariusz R. , Duszyński Jerzy , Evtodienko Yuri V.
Published: March 2003

The distribution of Ca2+ in intact cells was monitored with fluorescent probes: fura‐2 for cytosolic [Ca2+] and rhod‐2 for mitochondrial [Ca2+]. It was found that in neoplastic cells, such as Ehrlich ascites tumour and Zajdela hepatoma, but not in non‐malignant cells, such as fibroblasts, glucose and deoxyglucose elicited release of Ca2+ from endoplasmic reticulum stores and an increase in Ca2+ concentration in the cytosol. Parallel to this, a decrease in the rate of Ca2+ extrusion from the cell and an enhanced uptake of Ca2+ by mitochondria were observed. The increase in mitochondrial [Ca2+] was accompanied by an increase in the mitochondrial membrane potential and the reduction state of nicotinamide nucleotides. F1Fo‐ATPase in submitochondrial particles of Zajdela hepatoma was strongly inhibited in the presence of micromolar Ca2+ concentrations, whereas this activity in submitochondrial particles from rat liver appeared to be less sensitive to Ca2+. Indications of glycosylation of Ehrlich ascites tumour cell proteins were also obtained. These data strengthen the proposal [Bogucka, K., Teplova, V.V., Wojtczak, L. and Evtodienko, Y. V. (1995) Biochim. Biophys. Acta1228, 261–266] that the Crabtree effect is produced by mobilization of cell calcium, which is subsequently taken up by mitochondria and inhibits F1Fo‐ATP synthase.
Top‐down control analysis of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes
Authors: Ainscow Edward K. , Brand Martin D.
Published: March 2003

Control analysis was used to analyse the internal control of rat hepatocyte metabolism. The reactions of the cell were grouped into nine metabolic blocks linked by five key intermediates. The blocks were glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, mitochondrial proton leak, mitochondrial phosphorylation and ATP consumption. The linking intermediates were intracellular glucose‐6‐phosphate, pyruvate and ATP levels, cytoplasmic NADH/NAD ratio and mitochondrial membrane potential. The steady‐state fluxes through the blocks and the levels of the intermediates were measured in the absence and presence of specific effectors of hepatocyte metabolism. Application of the multiple modulation approach gave the kinetic responses of each block to each intermediate (the elasticities). These were then used to calculate all of the control coefficients, which describe the degree of control each block had over the level of each intermediate, and over the rate of each process. Within this full description of control, many different interactions could be identified. One key finding was that the processes that consumed ATP had only 35% of the control over the rate of ATP consumption. Instead, the reactions that produced ATP exerted the most control over ATP consumption rate; particularly important were mitochondrial phosphorylation (30% of control) and glycolysis (19%). The rate of glycolysis was positively controlled by the glycolytic enzymes themselves (66% of control) and by ATP consumption (47%). Mitochondrial production of ATP, including oxidative, proton leak and phosphorylation processes, had negative control over glycolysis (−26%; the Pasteur effect). In contrast, glycolysis had little control over the rate of ATP production by the mitochondria (−10%; the Crabtree effect). Control over the flux through the mitochondrial phosphorylation block was shared between pyruvate oxidation (23%), ATP consumption (28%) and the mitochondrial phosphorylation block itself (64%).
The efficient export of NADP‐containing glucose‐fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin‐arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding
Authors: Halbig Dirk , Wiegert Thomas , Blaudeck Natascha , Freudl Roland , Sprenger Georg A.
Published: March 2003

The periplasmic, NADP‐containing glucose‐fructose oxidoreductase of the gram‐negative bacterium Zymomonas mobilis belongs to a class of redox cofactor‐dependent enzymes which are exported with the aid of a signal peptide containing a so‐called twin‐arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose‐fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild‐type glucose‐fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose‐fructose oxidoreductase into the periplasm uses a Sec‐independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose‐fructose oxidoreductase is exported in a folded form. A glucose‐fructose oxidoreductase:β‐galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild‐type glucose‐fructose oxidoreductase expressed in trans but not of a typical Sec‐substrate (OmpA), indicating that the glucose‐fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.
Engineering of conformations of plasminogen activator inhibitor‐1
Authors: Kirkegaard Tove , Jensen Signe , Schousboe Susanne L. , Petersen Helle H. , Egelund Rikke , Andreasen Peter A. , Rodenburg Kees W.
Published: March 2003

The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface‐exposed reactive centre loop (RCL) is inserted as strand 4 in the large central β‐sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor‐1 (PAI‐1). We have investigated the importance of β‐strand 5A residues for the activity and latency transition of PAI‐1. An approximately fourfold increase in the rate of latency transition resulted from His‐substitution of Gln324 (position 334 in the α1‐proteinase inhibitor template numbering), which interacts with the underlying α‐helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting α‐helix F and β‐strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI‐1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild‐type PAI‐1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild‐type PAI‐1. We conclude that the anchoring of β‐strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes.
Evidence that phospholipase C‐γ2 interacts with SLP‐76, Syk, Lyn, LAT and the Fc receptor γ‐chain after stimulation of the collagen receptor glycoprotein VI in human platelets
Authors: Gross Barbara S. , Melford Steven K. , Watson Stephen P.
Published: March 2003

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor γ‐chain (FcR γ‐chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C‐γ2 (PLC‐γ2). In this study tyrosine‐phosphorylated proteins that associate with PLC‐γ2 after stimulation by a collagen‐related peptide (CRP) were characterized using glutathione S‐transferase fusion proteins of PLC‐γ2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC‐γ2. The majority of the tyrosine‐phosphorylated proteins that associate with PLC‐γ2 bind to its C‐terminal SH2 domain. These were found to include PLC‐γ2, Syk, SH2‐domain‐containing leucocyte protein of 76 kDa (SLP‐76), Lyn, linker for activation of T cells (LAT) and the FcR γ‐chain. Direct association was detected between PLC‐γ2 and SLP‐76, and between PLC‐γ2 and LAT upon CRP stimulation of platelets by far‐Western blotting. FcR γ‐chain and Lyn were found to co‐immunoprecipitate with PLC‐γ2 as well as with unidentified 110‐kDa and 75‐kDa phosphoproteins. The absence of an in vivo association between Syk and PLC‐γ2 in platelets is in contrast with that for PLC‐γ1 and Syk in B cells. The in vivo function of PLC‐γ2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C‐terminal SH2 domain is involved in the regulation of PLC‐γ2. These data indicate that the C‐terminal SH2 domain of PLC‐γ2 is important for PLC‐γ2 regulation through possible interactions with SLP‐76, Syk, Lyn, LAT and the FcR γ‐chain.
Acetylene hydratase of Pelobacter acetylenicus
Authors: Meckenstock Rainer U. , Krieger Robert , Ensign Scott , Kroneck Peter M. H. , Schink Bernhard
Published: March 2003

Acetylene hydratase of Pelobacter acetylenicus is a tungsten iron–sulfur protein involved in the fermentation of acetylene to ethanol and acetate. Expression of the enzyme was increased 10‐fold by feeding a 50‐L batch culture continuously with 104 Pa acetylene at pH 6.8–7.0. Acetylene hydratase was purified to homogeneity by a three‐step procedure in either the absence or presence of dioxygen. The enzyme was a monomer with a molecular mass of 73 kDa (SDS/PAGE) or 83 kDa (matrix‐assisted laser‐desorption ionization MS) and contained 0.5 ± 0.1 W (inductively coupled plasma/MS) and 1.3 ± 0.1 molybdopterin–guanine dinucleotide per mol. Selenium was absent.

 EPR spectra (enzyme as isolated, under air) showed a signal typical of a [3Fe–4S] cluster with gav = 2.01, at 10 K. In enzyme prepared under N2/H2, this signal was absent and reaction with dithionite led to a rhombic signal with gz = 2.048, gy = 1.939 and gx = 1.920 indicative of a low‐potential ferredoxin‐type [4Fe–4S] cluster. Upon oxidation with hexacyanoferrate(III), a new signal appeared with gx = 2.007, gy = 2.019 and gz = 2.048 (gav = 2.022), which disappeared after further oxidation. The signal was still visible at 150 K and was tentatively assigned to a W(V) center.

 The iron–sulfur center of acetylene hydratase (prepared under N2/H2) gave a midpoint redox potential of −410 ± 20 mV in a spectrophotometric titration with dithionite.

 Enzyme activity depended on the redox potential of the solution, with 50% of maximum activity at −340 ± 20 mV.

 The presence of a pterin–guanine dinucleotide cofactor differentiates acetylene hydratase from the aldehyde ferredoxin oxidoreductase‐type enzymes which have a pterin mononucleotide cofactor.
Effect of the ionic environment on the molecular structure of bacteriophage SPP1 portal protein
Authors: Jekow Petra , Behlke Joachim , Tichelaar Willem , Lurz Rudi , Regalla Manuela , Hinrichs Winfried , Tavares Paulo
Published: March 2003

Bacteriophage SPP1 portal protein is a large cyclical homo‐oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high‐point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13‐mers, named gp6H, at univalent salt concentrations in the hundred millimolar range (≥ 250 mm NaCl) or in the presence of bivalent cations in the millimolar range (≥ 5 mm MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape‐dependent frictional ratio, and a higher rate of intersubunit cross‐linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mm, the 13‐mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13‐mer, but maintained a defined tertiary structure. The association–dissociation equilibrium was mainly between the monomer and the 13‐mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10–50 mm) probably promotes complete association of gp6 into 13‐mer rings with a compact conformation.
The mature size of rat 4‐aminobutyrate aminotransferase is different in liver and brain
Authors: Kontani Yasuhide , Sakata Shigeko F. , Matsuda Koichi , Ohyama Tomoko , Sano Keiji , Tamaki Nanaya
Published: March 2003

The amino acid sequence predicted from a rat liver cDNA library indicated that the precursor of β‐AlaAT I (4‐aminobutyrate aminotransferase, β‐alanine–oxoglutarate aminotransferase) consists of a mature enzyme of 466 amino acid residues and a 34‐amino acid terminal segment, with amino acids attributed to the leader peptide. However, the mass of β‐AlaAT I from rat brain was larger than that from rat liver and kidney, as assessed by Western‐blot analysis, mass spectroscopy and N‐terminal sequencing. The mature form of β‐AlaAT I from the brain had an ISQAAAK‐ peptide on the N‐terminus of the liver mature β‐AlaAT I. Brain β‐AlaAT I was cleaved to liver β‐AlaAT I when incubated with fresh mitochondrial extract from rat liver. These results imply that mature rat liver β‐AlaAT I is proteolytically cleaved in two steps. The first cleavage of the motif XRX(↓)XS is performed by a mitochondrial processing peptidase, yielding an intermediate‐sized protein which is the mature brain β‐AlaAT I. The second cleavage, which generates the mature liver β‐AlaAT I, is also carried out by a mitochondrial endopeptidase. The second peptidase is active in liver but lacking in brain.
Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms
Authors: Burlacu‐Miron Simona , Gilles Anne‐Marie , Popescu Aurel , Bârzu Octavian , Craescu Constantin T.
Published: March 2003

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five‐stranded parallel β‐sheet surrounded by α‐helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long‐term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double‐labeled (13C and 15N) samples and double‐ and triple‐resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cβ resonances for more than 40% of the side chains in the free protein. Analysis of a 15N‐labeled enzyme in complex with the bi‐substrate analogue [P1,P5‐bis(5′‐adenosine)‐pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short‐range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the β‐strands in the unliganded form. The α‐helices and the β‐strands of the parallel β‐sheet in solution have the same limits (± 1 residue) as those observed in the crystal. The first helix (α1) appears to have a frayed N‐terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel β‐strands. The secondary structure of the nucleoside‐bound form, as deduced from intramolecular NOEs and the 1Hα chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein–ligand contacts. 1H/2H exchange experiments performed on free and Ap5A‐bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N‐side of the parallel β‐sheet.
The amino‐terminal region of the Escherichia coli T‐protein of the glycine cleavage system is essential for proper association with H‐protein
Authors: Okamura‐Ikeda Kazuko , Fujiwara Kazuko , Motokawa Yutaro
Published: March 2003

T‐protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate‐dependent reaction. Our previous work on Escherichia coli T‐protein (ET) showed that the lack of the N‐terminal 16 residues caused a loss of catalytic activity [Okamura‐Ikeda, K., Ohmura, Y., Fujiwara, K. and Motokawa, Y. (1993) Eur. J. Biochem.216, 539–548]. To define the role of the N‐terminal region of ET, a series of deletion mutants were constructed by site‐directed mutagenesis and expressed in E. coli. Deletions of the N‐terminal 4, 7 and 11 residues led to reduction in the activity to 42, 9 and 4%, respectively, relative to the wild‐type enzyme (wtET). The mutant with 7‐residue deletion (ETΔ7) was purified and analyzed. ETΔ7 exhibited a marked increase in Km (25‐fold) for E. coli H‐protein (EH) accompanied by a 10‐fold decrease in kcat compared with wtET, indicating the importance of the N‐terminal region in the interaction with EH. The role of this region in the ET–EH interaction was investigated by cross‐linking of wtET–EH or ETΔ7–EH complex with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, a zero‐length cross‐linker, in the presence of folate substrates. The resulting tripartite cross‐linked products were cleaved with lysylendopeptidase and V8 protease. After purification by reversed‐phase HPLC, the cross‐linked peptides were subjected to Edman sequencing. An intramolecular cross‐linking between Asp34 and Lys216 of wtET which was not observed in wtET alone and an intermolecular cross‐linking between Lys288 of wtET and Asp‐43 of EH were identified. In contrast, no such cross‐linking was detected from the cross‐linked product of ETΔ7. These results suggest that EH, when it interacts with ET, causes a change in conformation of ET and that the N‐terminal region of ET is essential for the conformational change leading to the proper interaction with EH.
CD and NMR investigations on trifluoroethanol‐induced step‐wise folding of helical segment from scorpion neurotoxin
Authors: Khandelwal Purnima , Seth Subhendu , V. Hosur Ramakrishna
Published: March 2003

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2‐Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly α‐helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step‐wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5–11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.
Structural and kinetic studies of the pyruvate–ferredoxin oxidoreductase/ferredoxin complex from Desulfovibrio africanus
Authors: Pieulle Laetitia , Charon Marie‐Hélène , Bianco Pierre , Bonicel Jacques , Pétillot Yves , Hatchikian E. Claude
Published: March 2003

The pyruvate–ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Desulfovibrio africanus has been investigated with the aim of understanding more fully protein–protein interaction and the kinetic characteristics of electron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron acceptors for PFOR. The complete amino acid sequence of Fd II was determined by automatic Edman degradation. It revealed a striking similarity to that of Fd I. The protein consists of 64 residues and its amino acid sequence is in agreement with a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II contains five cysteine residues of which the first four (Cys11, Cys14, Cys17 and Cys54) are likely ligands for the single [4Fe–4S] cluster. A covalently cross‐linked complex between PFOR and Fd I or Fd II was obtained by using a water soluble carbodiimide. This complex exhibited a stoichiometry of one ferredoxin for one PFOR subunit and is dependent on the ionic strength. The second‐order rate constants for electron transfer between PFOR and Fds determined electrochemically using cyclic voltammetry are 7 × 107 m−1·s−1 for Fd I and 2 × 107 m−1·s−1 for Fd II and Fd III. The Km values of PFOR for Fd I and Fd II measured both by the electrochemical and the spectrophotometric method have been found to be 3 µm and 5 µm, respectively. The three‐dimensional modelling of Fd II and surface analysis of Fd I, Fd II and PFOR suggest that a protein–protein complex is likely to be formed between aspartic acid/glutamic acid invariant residues of Fds and lysine residues surrounding the distal [4Fe–4S] cluster of PFOR. All of these studies are indicative of the involvement of electrostatic interactions between the two redox partners.
Type‐II 3‐oxoacyl‐CoA thiolase of the nematode Caenorhabditis elegans is located in peroxisomes, highly expressed during larval stages and induced by clofibrate
Authors: Maebuchi Motohiro , Togo Summanuna H. , Yokota Sadaki , Ghenea Simona , Bun‐ya Masanori , Kamiryo Tatsuyuki , Kawahara Akira
Published: March 2003

We examined the expression and localization of type‐II 3‐oxoacyl‐CoA thiolase in the nematode Caenorhabditis elegans. Type‐II thiolase acts on 3‐oxoacyl‐CoA esters with a methyl group at the α carbon, whereas conventional thiolases do not. Mammalian type‐II thiolase, which is also termed sterol carrier protein x (SCPx) or SCP2/3‐oxoacyl‐CoA thiolase, is located in the peroxisomes and involved in phytanic acid degradation and most probably in bile acid synthesis. The nematode enzyme lacks the SCP2 domain, which carries the peroxisomal‐targeting signal, but produces bile acids in a cell‐free system. Northern and Western blot analyses demonstrated that C. elegans expressed type‐II thiolase throughout its life cycle, especially during the larval stages, and that the expression was significantly enhanced by the addition of clofibrate at 5 mm or more to the culture medium. Whole‐mount in situ hybridization and immunostaining of L4 larvae revealed that the enzyme was mainly expressed in intestinal cells, which are multifunctional like many of the cell types in C. elegans. Subcellular fractionation and indirect immunoelectron microscopy of the nematode detected the enzyme in the matrix of peroxisomes. These results indicate the fundamental homology between mammalian SCPx and the nematode enzyme regardless of whether the SCP2 part is fused, suggesting their common physiological roles.
Inhibition of human immunodeficiency virus (HIV‐1) replication in SupT1 cells transduced with an HIV‐1 LTR‐driven PKR cDNA construct
Authors: Adelson Martin E. , Martinand‐Mari Camille , Iacono Kathryn T. , Muto Nicholas F. , Suhadolnik Robert J.
Published: March 2003

Current strategies against the human immunodeficiency virus type 1 (HIV‐1), including nucleoside analogues and protease inhibitors, have limited effectiveness as shown by the evolution of resistant retroviral strains and the presence of latent HIV‐1 reservoirs. Therefore, it is necessary to look beyond anti‐retroviral strategies and to rely on the body’s immune system to inhibit HIV‐1 replication. In this study, we approach the inhibition of HIV‐1 replication by upregulation of the antiviral pathway that is natural to mammalian cells. Vectors were constructed which were capable of transferring the antiviral enzyme, p68 kinase (PKR), into target SupT1 lymphoblastoid cells under HIV‐1 LTR transcriptional regulation via a retroviral‐mediated shuttle system. We report a significant inhibition of HIV‐1 replication in HIV‐1 LTR‐PKR cDNA transduced clones (105–10 : 239 and 106–4 : 560) expressing different PKR levels as measured by inhibition of HIV‐1 induced syncytia formation and HIV‐1 reverse transcriptase activity. Whereas the expression of PKR in parental vector transduced clone (N2–20P) is down‐regulated 48 h after HIV‐1 infection, the two transduced clones (one with PKR in the forward orientation and one in the reverse orientation) demonstrate increased PKR expression through 96 h post‐infection, concomitant with an increase in eIF‐2α phosphorylation and an increase in NF‐κB activity at 72 h postinfection. These results demonstrate that the overexpression of PKR can inhibit HIV‐1 replication and confirm the involvement of PKR in the IFN‐associated antiviral pathway against HIV‐1 infection. Finally, the treatment of the transduced clone 106–4 : 560 with AZT resulted in complete inhibition of HIV‐1 replication.
The 24‐kDa iron–sulphur subunit of complex I is required for enzyme activity
Authors: Almeida Teresa , Duarte Margarida , Melo Ana M. P. , Videira Arnaldo
Published: March 2003

We have cloned the nuclear gene encoding the 24‐kDa iron–sulphur subunit of complex I from Neurospora crassa. The gene was inactivated in vivo by repeat‐induced point‐mutations, and mutant strains lacking the 24‐kDa protein were isolated. Mutant nuo24 appears to assemble an almost intact complex I only lacking the 24‐kDa subunit. However, we also found reduced levels of the NADH‐binding, 51‐kDa subunit of the enzyme. Surprisingly, the complex I from the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreement with this, the respiration of intact mitochondria or mitochondrial membranes from the mutant strain is insensitive to rotenone inhibition. These results suggest that the nuo24 complex is not functioning in electron transfer and the 24‐kDa protein is absolutely required for complex I activity. This phenotype may explain the findings that the 24‐kDa iron–sulphur protein is reduced or absent in human mitochondrial diseases. In addition, selected substitutions of cysteine to alanine residues in the 24‐kDa protein suggest that binding of the iron–sulphur centre is a requisite for protein assembly.
The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity
Authors: Chenal‐Francisque Viviane , Tourneux Lise , Carniel Elisabeth , Christova Petya , de la Sierra Inès Li , Bârzu Octavian , Gilles Anne‐Marie
Published: March 2003

Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in α‐helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the two enzymes was related to their ability to phosphorylate 3′‐deoxy‐3′‐azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three‐dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation.
Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age‐dependent role change of the worker honeybee (Apis mellifera L.)
Authors: Ohashi Kazuaki , Natori Shunji , Kubo Takeo
Published: March 2003

Worker honeybees change their behaviour from the role of nurse to that of forager with age. We have isolated cDNA clones for two honeybee (Apis mellifera L.) genes, encoding α‐amylase and glucose oxidase homologues, that are expressed in the hypopharyngeal gland of forager bees. The predicted amino acid sequence of the putative Apis amylase showed 60.5% identity with Drosophila melanogasterα‐amylase, whereas that of Apis glucose oxidase showed 23.8% identity with Aspergillus niger glucose oxidase. To determine whether the isolated cDNAs actually encode these enzymes, we purified amylase and glucose oxidase from homogenized forager‐bee hypopharyngeal glands. We sequenced the N‐terminal regions of the purified enzymes and found that they matched the corresponding cDNAs. mRNAs for both enzymes were detected by Northern blotting in the hypopharyngeal gland of the forager bee but not in the nurse‐bee gland. These results clearly indicate that expression of the genes for these carbohydrate‐metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age‐dependent role change of the worker.
LIS1 is a microtubule‐associated phosphoprotein
Authors: Sapir Tamar , Cahana Aviv , Seger Rony , Nekhai Sergei , Reiner Orly
Published: March 2003

Lissencephaly, a severe brain malformation, may be caused by mutations in the LIS1 gene. LIS1 encodes a microtubule‐associated protein (MAP) that is also part of the enzyme complex, platelet‐activating factor acetylhydrolase. LIS1 is also found in a complex with two protein kinases; a T‐cell Tat‐associated kinase, which contains casein‐dependant kinase (CDK) activating kinase (CAK), as well as CAK‐inducing activity, and with a spleen protein‐tyrosine kinase similar to the catalytic domain of p72syk. As phosphorylation is one of the ways to control cellular localization and protein–protein interactions, we investigated whether LIS1 undergoes this post‐translational modification. Our results demonstrate that LIS1 is a developmentally regulated phosphoprotein. Phosphorylated LIS1 is mainly found in the MAP fraction. Phosphoamino acid analysis revealed that LIS1 is phosphorylated on serine residues. Alkaline phosphatase treatment reduced the number of visible LIS1 isoforms. In‐gel assays demonstrate a 50‐kDa LIS1 kinase that is enriched in microtubule‐associated fractions. In vitro, LIS1 was phosphorylated by protein kinase CKII (casein kinase II), but not many other kinases that were tested. We suggest that LIS1 activity may be regulated by phosphorylation.
Irreversible site‐directed labeling of the 4‐aminobutyrate binding site by tritiated meta‐sulfonate benzene diazonium
Authors: Jacques Patrice , Perret Philippe , Bouchet Marie‐Jeanne , Foucaud Bernard , Goeldner Maurice , Benke Dietmar
Published: March 2003

Tritiated meta‐sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4‐aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45–60 kDa molecular mass range which were identified as α1 subunits and β2/β3 subunits by immunoprecipitation with subunit‐specific antibodies. The specific immunoprecipitation of α and β subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the β subunit but reveal also the contribution of yet unidentified nucleophilic residues on the α subunit for the GABA binding site.
Placenta‐specific transcripts of the aromatase encoding gene include different untranslated first exons in sheep and cattle
Authors: Vanselow Jens , Zsolnai Attila , Fésüs Laszlo , Fürbass Rainer , Schwerin Manfred
Published: March 2003

The aim of the present study was the characterization of the ovine aromatase cytochrome P450 encoding gene (Cyp19) and the analysis of its tissue‐specific expression. Two loci with considerable sequence identity were found (Cyp19 and Cyp19b). From Cyp19, tissue‐specific transcript variants with different untranslated first exons but identical coding regions could be identified. Cyp19b transcripts were not detected. In the sheep brain and ovarian granulosa cells transcript variants, starting with the untranslated exons 1.4 and 2, respectively, were preferentially found. Exons 1.2 and 1.3 which had been described in bovines could not be detected in sheep and the major 5′ untranslated region of the bovine placental transcript, exon 1.1, was also not found to predominate in the sheep placenta. However this exon frequently was combined with a new untranslated exon (exon 1.1a) thus generating an alternative splice variant. The main placental transcripts in sheep had a different first exon (exon 1.5). Two alternatively spliced variants of this transcript were found with tissue‐specific preference.

 From the present data it can be concluded: (a) that the ovine genome contains two copies of Cyp19 of which only one is transcribed and may encode a functional protein; and (b) that in spite of being closely related species, sheep and cattle have remarkable differences concerning tissue‐specific transcript distribution and presumable promoter usage.
Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri
Authors: Meuer Jörn , Bartoschek Stefan , Koch Jürgen , Künkel Andreas , Hedderich Reiner
Published: March 2003

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane‐bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme‐iron and 10.8 mol of acid‐labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe‐4S] ferredoxin with a specific activity of 50 U·mg protein−1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 µm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U·mg protein−1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 µm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 µm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate‐grown, methanol‐grown or H2/CO2‐grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin‐linked H2 production coupled to the oxidation of the carbonyl‐group of acetyl‐CoA to CO2 during growth on acetate, and to ferredoxin‐linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.
Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline
Authors: Delmotte Caroline , Le Guern Emmanuelle , Trudelle Yves , Delmas Agnès
Published: March 2003

Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT‐NP6 chimeric peptide, consisting of two copies of a promiscuous T‐helper epitope (T: residues 288–302 from the fusion protein of the measles virus) linked to the NP6 T‐cytotoxic epitope (NP6: residues 52–60 from the nucleoprotein of measles virus) was able to induce virus‐specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT‐NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self‐associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal‐dependence studies show that the unfolding of self‐associated α‐helices is significantly more pronounced than the unfolding of isolated α‐helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT‐NP6 peptide.
The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core
Authors: Mei Giampiero , Di Venere Almerinda , Campeggi Flaminia Malvezzi , Gilardi Gianfranco , Rosato Nicola , De Matteis Fabio , Finazzi‐Agrò Alessandro
Published: March 2003

The unfolding of the blue‐copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two‐state model. The total free energy change from the native to the unfolded state was 9.4 ± 0.4 kcal·mol−1, while a lower value (6.4 ± 0.4 kcal·mol−1) was obtained for the metal depleted enzyme (apo‐azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo‐azurin were practically unaffected by hydrostatic pressure up to 3000 bar.

 Site‐directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 ± 0.3 kcal·mol−1 and 4.8 ± 0.3 kcal·mol−1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild‐type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady‐state fluorescence measurements. The change in volume associated to the pressure‐induced unfolding was estimated to be −24 mL·mol−1 for Ile7Ser and −55 mL·mol−1 for Phe110Ser.

 These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.
HSF3 is a major heat shock responsive factor duringchicken embryonic development
Authors: Kawazoe Yoshinori , Tanabe Masako , Sasai Noriaki , Nagata Kazuhiro , Nakai Akira
Published: March 2003

The expression of heat shock genes in vertebrates is regulated mainly at the level of transcription by four heat shock transcription factors (HSFs 1–4). Avian cells express at least three HSFs (HSFs 1–3). HSF1 is rapidly activated by even mild heat shock, while HSF3 is activated only by severe heat shock. In contrast, HSF2 is not activated by heat stress and has been speculated to have developmental functions. Here, we examined the temporal and spatial profiles of changes in the levels of these three HSFs in various tissues during chicken development. We found that HSF3 was almost constantly expressed in various tissues during early to late chicken development. The expression of HSF1 was equally high in most tissues early in development and thereafter declined to different levels in a tissue‐dependent manner. Thus, HSF3 became the dominant heat‐responsive factor in all tissues examined. The magnitude of heat shock response determined by Northern blotting did not always correlate with the level of HSF1 expression, suggesting that not only HSF1 but also HSF3 may be a major factor mediating stress signals to heat shock gene expression in the chicken. In addition, the high‐level and ubiquitous expression of HSF2 as well as HSF1 and HSF3 in early embryogenesis suggested the involvement of these factors in all developmental processes.