bluuebunny/crossref_metadata_2025 huggingface dataset:
Explicit Constants for the Dextran‐Sulfate‐Mediated Activation and Autoactivation of Purified Human Factor XII (Hageman Factor)
Authors: Loiseau Céline , Randriamahazaka Hyacinthe N. , Nigretto Jean‐Maxime
Published: July 2004

The autoactivation kinetics of purified factor XII (FXII) in the presence of dextran sulfate of 500 000 Da was reexamined assuming the existence of two preceding activation steps. Kinetics were numerically simulated by using rate and equilibrium constants related to surface‐bound species. Relevant feature parameters related to the polymer (number of binding sites and concentration, dissociation constant of FXII from the surface) and the zymogen (concentration, Michaelis‐Menten constant of the autoactivation reaction, catalytic rate constant) were accordingly introduced in the mechanisms. Depending on the rate‐limiting step, i.e. whether the polymer or FXII predominates, numerical simulation analysis led to obtain for the observed autoactivation rate constant (kobs) two explicit expressions which included the contributing variables.

One of the two proposed models was in good accordance with the experimental data obtained in this study and with others published previously. We were able to estimate the mean number of the FXII‐activating sites supported by the polymer chains (220) and the equilibrium dissociation constant of FXII from the surface (1 μM). Further treatment led us to determine surface‐concentration‐independant constants (Km= 2510 nM and kcat= 0.01 s‐1), as well as the rate constant (k1= 1.6×10‐4 s‐1) of the postulated first‐order activation rate aimed at explaining the formation of the first trace amounts of FXIIa via an intramolecular mechanism. Overall, the treatment applied to the dextran sulfate case offers a quantitative tool by which data determined in the presence of other activating materials can be rationalized.
Membrane‐Bound c ‐Type Cytochromes in Heliobacillus mobilis
Authors: Nitschke Wolfgang , Schoepp Barbara , Floss Barbara , Schricker Anna , Rutherford A. William , Liebl Ursula
Published: July 2004

The spectral and electrochemical parameters, as well as the orientations of the heme planes with respect to the membrane plane, of the c ‐type hemes present in membrane fragments from Heliobacillus mobilis were characterised by optical and EPR spectroscopy. Cytochrome c553 was thereby shown to represent at least four and possibly five heme species with the following characteristics: Em= ‐60 mV± 10 mV, gz= 2.92, 60°; Em=+ 90 mV ± 10 mV, gz= 2.92, 90°; Em=+ 120 mV ± 20 mV, gz= 3.03; and Em=+ 170 mV ± 20 mV, gz= 3.03. The latter component may correspond to two hemes with redox midpoint potentials of Em=+160 mV ± 20 mV and Em=+180 mV ± 20 mV (all Em values at pH 7.0). For the heme species having gz peaks at g≈3.03, determination of individual orientations was precluded due to the superposition of several differently oriented hemes. About one copy of each heme was found to be present per photosynthetic reaction centre, with the exception of the +120 mV component for which a stoichiometry of 2 hemes/reaction centre was obtained. The heme proteins were detergent‐solubilised and partially purified. Three c ‐type cytochromes that migrated with apparent molecular masses of 18, 29 and 50 kDa were detected on SDS/PAGE. Optical redox titrations at pH 7.0 showed redox midpoint potentials of +160 mV ± 10 mV for the 18‐kDa cytochrome, and ‐60 mV ± 10 mV, with possible contributions around +160 mV, for the 50‐kDa cytochrome. A tentative attribution of heme species observed in membranes to the isolated heme proteins is presented. The results obtained on H. mobilis are compared with those reported for green sulphur bacteria.
Modulation of the Activity of the Human Cholesteryl Ester Transfer Protein by Carboxylated Derivatives
Authors: Florentin Emmanuel , Athias Anne , Lacrost Laurent
Published: July 2004

The influence of palmitic acid, 13‐cis‐retinoic acid, all‐trans‐retinoic acid, and all‐trans‐retinol on the activity of the human cholesteryl ester transfer protein (CETP) was evaluated either in total human plasma supplemented with a tracer dose of 3H‐labeled cholesteryl‐ester‐containing high‐density lipoprotein sub‐fraction 3 ([3H]CE‐HDL3), or in reconstituted mixtures containing [3H]CE‐HDL3, isolated low‐density lipoproteins (LDL), and purified CETP. In reconstituted mixtures, all the carboxylated derivatives increased progressively and significantly the transfer of 3H‐labeled cholesteryl esters from [3H]CE‐HDL3 towards LDL in the 20–100 μM concentration range. Under identical experimental conditions, CETP activity was only minimally modified in the presence of all‐trans‐retinol. When present at a concentration of 60, 80, or 100 μM, 13‐cis‐retinoic acid was a significantly more potent activator of CETP activity than all the other derivatives studied (P < 0.01 in all cases). In contrast to observations made with reconstituted mixtures, only 13‐cis‐retinoic acid, but not palmitic acid, was able to induce a significant, concentration‐dependent stimulation of CETP activity in total human plasma. In fact, differences in the ability of 13‐cis‐retinoic acid and palmitic acid to modulate the plasma cholesteryl ester transfer reaction were linked to their relative affinity for albumin and lipoprotein substrates: fatty‐acid‐poor albumin reduced CETP activity to a significantly greater extent in reconstituted mixtures containing palmitic acid than in reconstituted mixtures containing 13‐cis‐retinoic acid (P < 0.01 for all the incubation mixtures in the 1–10 g/l albumin concentration range); palmitic acid presented a markedly lower ability to increase the electrophoretic mobility of LDL and HDL fractions in total plasma than 13‐cis‐retinoic acid. In support of a key role of the negatively charged carboxylic group of 13‐cis‐retinoic acid in upregulating CETP activity, cholesteryl ester transfer rates correlated positively with the electrophoretic mobility of LDL (r= 0.98: P < 0.0002) and HDL (r= 0.96; P < 0.0008) in total plasma supplemented with the carboxylated compound. It is concluded that 13‐cis‐retinoic acid can upregulate the CETP‐mediated cholesteryl ester transfer reaction both in reconstituted mixtures containing isolated lipoproteins and purified CETP, and in total normolipidemic human plasma.
Blockade of the α2‐Macroglobulin Receptor/Low‐Density‐Lipoprotein‐Receptor‐Related Protein on Rat Liver Parenchymal Cells by the 39‐kDa Receptor‐Associated Protein Leaves the Interaction of β‐Migrating very‐Low‐Density Lipoprotein with the Lipoprotein Remnant Receptor Unaffected
Authors: Ziere Gijsbertus J. , Van Der Kaaden Marieke E. , Vogelezang Carla J. M. , Boers Wim , Bihain Bernard E. , Kuiper Johan , Kruijt J. Kar , Van Berkel Theo J. C.
Published: July 2004

The nature of the liver binding site which is responsible for the initial recognition and clearance of chylomicron‐remnants and β‐migrating very‐low‐density lipoprotein (β‐VLDL) is under active dispute. We have investigated the effect of the 39‐kDa receptor‐associated protein (RAP) on the recognition site for activated α2‐macroglobulin and β‐VLDL on rat liver parenchymal cells in vivo and in vitro in order to analyze whether both substrates are recognized and internalized by the same receptor system. Radiola‐belled trypsin‐activated α2‐macroglobulin (α2M‐T) was cleared rapidly by the liver (maximal uptake of 80.8 ±1.0% of the injected dose). Prior injection of 5, 15, or 50 mg gluthathione‐S ‐transferase‐linked RAP (GST‐RAP)/kg rat reduced the liver uptake to 62.2 ±2.3%, 59.3 ± 1.1%, or 2.9 ± 0.1 % of the injected dose, respectively. Concurrently the serum decay was strongly delayed after injection of 50 mg GST‐RAP/kg rat but this did not affect the serum decay and liver uptake of 125I‐β‐VLDL. Binding studies with isolated liver parenchymal cells in vitro demonstrated that the binding of 125I‐α2M‐T was 98% inhibited by GST‐RAP with an IC50 of 0.3 μg/ml (4.2nM), whereas the binding of 125I‐β‐VLDL and 125I‐β‐VLDL + recombinant apolipoprotein E (rec‐apoE) was unaffected by GST‐RAP up to 50 μg/ml (700 nM). Also, the cell association and degradation of α2M‐T was blocked by RAP, while the association and degradation of β‐VLDL and β‐VLDL + rec‐apoE were not influenced. The inhibitory effect of RAP on the cell association and degradation of α2M‐T lasted for 1–2 h of incubation at 37°C. The binding of the radioiodinated RAP to isolated liver parenchymal cells was highly efficiently coupled to lysosomal degradation. Upon in vivo injection into rats, 125I‐labeled RAP is rapidly cleared from the serum and taken up by the liver, which is also coupled to efficient degradation. Since RAP blocks binding of all known ligands to the α2‐macroglobulin receptor/low‐density lipoprotein receptor‐related protein (the α2Mr/LRP) and at high concentrations the binding to the LDL receptor, we conclude that the initial binding and internalization of β‐VLDL by rat liver parenchymal cells is not mediated by the α2Mr/LRP. The properties of binding of β‐VLDL to rat liver parenchymal cells points to an apoE‐specific recognition site for lipoprotein remnants which differs from the α2Mr/LRP, proteoglycans and the LDL receptor and is tentatively called the lipoprotein remnant receptor.
Restoration of Clostridium Difficile Toxin‐B‐Inhibited Phospholipase D by Phosphatidylinositol 4,5‐Bisphosphate
Authors: Schmidt Martina , Rümenapp Ulrich , Nehls Christoph , Ott Sabine , Keller Jutta , Von Eichel‐Streiber Christoph , Jakobs Karl H.
Published: July 2004

Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins. Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), concomitant with a large increase in PtdIns(4,5)P2. On the other hand, neoinycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP[S]‐stimulated PLD activities. Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2–3‐fold, whereas various other phospholipids were ineffective. Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40%. In membranes of toxin‐B‐treated cells, basal and GTP[S]‐stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate. Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP[S]‐stimulated PLD activities in membranes of toxin‐B‐treated cells. In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho‐inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.
Conformational Analysis of Blood Group A Trisaccharide in Solution and in the Binding Site of Dolichos biflorus Lectin Using Transient and Transferred Nuclear Overhauser Enhancement (NOE) and Rotating‐Frame NOE Experiments
Authors: Casset Florence , Peters Thomas , Etzler Marilynn , Korchagina Elena , Nifant'ev Nikolai , Pérez Serge , Imberty Anne
Published: July 2004

The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (α‐d‐GalNAc(l→3)[α‐d‐Fuc(l→2)]β‐d‐Gal‐O ‐R) in the combining site of Dolichos biflorus seed lectin by use of 400‐MHz and 600‐MHz NMR spectroscopy. D. biflorus lectin displays a unique specificity for CalNAc residues. It occurs in solution as a tetranieric assembly having a molecular mass of 110kDa, with two carbohydrate‐binding sites per molecule. First, NOE build‐up curves were obtained for the free blood group A trisaccharide from one‐dimensional transient NOE experiments. Simulated NOE build‐up curves were constructed from an ensemble of low‐energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low‐energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two‐dimensional transferred NOE and rotating‐frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating‐frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build‐up curves were compared to theoretical curves calculated for both low‐energy conformers located in the D. biflorus lectin‐binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein‐ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. biflorus lectin.
In vitroα‐Complementation of β‐Galactosidase on a Bacteriophage Surface
Authors: Dunn Ian S.
Published: July 2004

Surface display of large multimeric non‐secreted proteins is advantageous on the bacteriophage λ compared with the widely used filamentous phage systems. A model system, the α‐complementation of β‐galactosidase, was used for both further general characterization of protein‐protein interactions on the λ tail tube surface and for specifically probing the structure of the phage‐displayed β‐galactosidase tetramer. In this complementation system, dimeric enzymatically inactive N‐terminal deletion mutants of β‐galactosidase (α‐acceptors) interact with peptides whose sequences span the region of the deletion (α‐peptides) with the subsequent formation of tetramers and restoration of activity. The λ phage could tolerate incorporation into their tail tubes of a limited number of copies of V protein (gpV) subunits C‐terminally modified with an active α‐peptide. Purified α‐peptide phage showed specific in vitroα‐complementation with an α‐acceptor extract; the features of this reaction suggested that each complemented monomer can directly associate with an α‐peptide displayed within the same tail tube structure. In contrast to the α‐peptide, attempts to surface display an α‐acceptor protein in a similar manner were unsuccessful. The implications of this work for surface‐display cDNA libraries are discussed.
A Conserved Sequence Region of Scorpion Toxins Rendered Immunogenic Induces Broadly Cross‐Reactive, Neutralizing Antibodies
Authors: Devaux Christiane , Fourquet Patrick , Granier Claude
Published: July 2004

Scorpion toxins contitute a family of proteins with a high degree of sequence diversity but a common mode of action. Neutralization of the toxic effects of scorpion stings by serotherapy is limited due to the various serotypes expressed by these proteins. We explored the possibility of raising antibodies to conserved parts of the toxins which could recognize several members of the family. We established the variability profile of a set of 25 scorpion toxin sequences, then evaluated systematically by peptidescanning methods the antigenicity of one scorpion toxin. The most conserved regions were generally very poorly antigenic. One exception was the N‐terminal region, which is both conserved and antigenic. Antibodies were raised in rabbits against an eight‐residue synthetic peptide mimicking the N‐terminal region. These peptide antibodies were cross‐reactive with several scorpion toxins belonging to different serotypes and neutralized both the pharmacological effects (binding to rat brain synaptosomes) and the biological activity (toxicity in mice) of the parent toxin. The molecular model of the toxin indicates that antibody binding to residues 1–8 probably either masks some residue(s) of the N‐terminus critical for the biological activity or overlaps with the epitope previously defined by neutralizing monoclonal antibody. These findings could open the way for new therapeutic strategies for the medical care of envenomations.
Alternative Processing of the Tryptophanyl‐tRNA Synthetase mRNA from Interferon‐Treated Human Cells
Authors: Turpaev Kyril T. , Zakhariev Vladimir M. , Sokolova Irina V. , Narovlyansky Alexander N. , Amchenkova Aleftina M. , Justesen Just , Frolova Lyudmila Yu.
Published: July 2004

We have analysed the structure of mRNA isoforms of the human gene encoding tryptophanyl‐tRNA synthetase (Trp‐tRNA synthetase) expressed in the epithelial CaOv cells and MT‐4 lymphocytes. The Trp‐tRNA synthetase gene is induced by interferon‐γ in both lines and, in MT‐4 lymphocytes, also by interferon‐α. Four Trp‐tRNA synthetase mRNA isoforms have different combinations of the first exons IA, IB and II. Two transcription initiation sites (P1 and P2) were detected 90 bp from each other. Processing of the primary transcript initiated from the PI start site generates the mRNA isoform where exon IA joins to exon II. The other three isoforms are produced by alternative splicing of the primary transcript produced from the P2 start site. Isoform 2 has a 3′‐end fragment of exon IA joined to exon II. Isoform 3 contains exons IA and IB. Isoform 4 contains exon IA and exon III and lacks exon II encoding the N‐terminus of the Trp‐tRNA synthetase. Therefore, the two primary transcripts of the Trp‐tRNA synthetase gene differ only in the 5′ flank sequence between P1 and P2, and this fragment regulates their processing. Both interferon‐α and interferon‐γ induce exon IA‐containing and exon IB‐containing isoforms of the Trp‐tRNA synthetase mRNA.
Ser57 in the Na+/Proline Permease of Escherichia coli is Critical for High‐Affinity Proline Uptake
Authors: Quick Matthias , Tebbe Sandra , Jung Heinrich
Published: July 2004

Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild‐type value. In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs l‐azetidine‐2‐carboxylate and 3,4‐dehydro‐d,l‐proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18, respectively, relative to wild‐type. In contrast, all of the Ser57 replacements analyzed cause only small changes in Vmax values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild‐type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate‐binding domain of the protein.
Anaerobic Crystallisation of an Isopenicillin N Synthase · Fe(II) · Substrate Complex Demonstrated by X‐Ray Studies
Authors: Roach Peter L. , Clifton Ian J. , Hensgens Charles M. H. , Shibata Norio , Long Alexandra J. , Strange Richard W. , Hasnain Samar S. , Schofield Christopher J. , Baldwin Jack E. , Hajdu Janos
Published: July 2004

Isopenicillin N synthase (IPNS) was cocrystallised with ferrous sulphate and its substrate, δ‐(L‐α‐aminoadipoyl)‐L‐cysteinyl‐D‐valine (Aad‐Cys‐Val). Vital to the successful procedure was the maintenance of a rigorously anaerobic environment. Hanging‐drop vapour‐diffusion crystallisation experiments, using lithium sulphate as the precipitant produced three crystal forms. Form I crystals, with a plate habit, diffracted X‐rays to at least 0.11‐nm resolution at the European Synchrotron Radiation Facility and belong to the space group P212121, with unit‐cell dimensions a= 4.68, b= 7.15, c= 10.10 nm. Their asymmetric unit contains a single IPNS. Fe(II). Aad‐Cys‐Val complex with a solvent content of 38.5%. Form II crystals, with a hexagonal habit, diffract X‐rays to at least 0.21 nm resolution at the European Synchrotron Radiation Facility and belong to the space group P3121, with unit‐cell dimensions a= 10.10, b= 10.10, c= 11.567 nm. Their asymmetric unit also contains a single IPNS. Fe(II). Aad‐Cys‐Val complex with a solvent content of 69.5%. Form III crystals, needles, do not show well‐ordered diffraction. Although all three forms were initially produced in crystallisation experiments under identical conditions, appropriate micro and streak seeding allows selective crystallisation of form I or form II crystals. Extended X‐ray‐absorption fine‐structure studies on a crystalline slurry of the form I crystals demonstrate the presence of an Fe‐S(Aad‐Cys‐Val) bond length of 0.234±20.003 nm.
Primary Structure and Characterization of an Exopolygalacturonase from Aspergillus Tubingensis
Authors: Kester Harry C. M. , Someren Margo A. Kusters‐Van , Müller Yvonne , Visser Jaap
Published: July 2004

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH‐range 3.7–4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4‐α‐d‐galacturonide)galacturonohydrolase] that cleaves monomer units from the non‐reducing end of the substrate molecule.

K
m and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml−1 and 3.1 mg ml−1 and 255 U mg I and 262 U mg−1 for the wild‐type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2–7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2–7), the Michaelis constant (Km) decreased with increasing chain length (n). The Vmax value increased with chain length up to n= 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki= 0.3 mM) as well as by reduced digalacturonate (Ki= 0.4 mM).

The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX‐coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.
Mathematical Modelling of the Citric Acid Cycle for the Analysis of Glutamine Isotopomers from Cerebellar Astrocytes Incubated with [1‐13C]glucose
Authors: Merle Michel , Martin Magali , Villégier Annick , Canioni Paul
Published: July 2004

A mathematical model of the citric acid cycle devoted to the analysis of 13C‐NMR data was developed for determining the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. Different variants of the model were considered depending on the reversibility of the conversion of fumarate into malate and oxaloacetate. The model also included the possibility of orientation‐conserved transfer of the four‐carbon citric acid cycle intermediates, leading to conversion of succinyl‐CoA C1 into either malate C1 or C4. It was used to analyse NMR data from glutamine isotopomers produced by cerebellar astrocytes incubated with [1‐13C]glucose. Partial cycling (39%) between oxaloacetate and fumarate was evident from the analysis. Application of the model to glutamate isotopomers from granule cells incubated with [1‐13C]glucose [Martin, M., Portais, J. C., Labouesse, J., Canioni, P. & Merle, M. (1993) Eur. J. Biochem. 217, 617–625] indicated that total cycling of oxaloacetate into fumarate was, in this case, required to get the best fit. The results emphasized some important differences in carbon metabolism between cerebellar astrocytes and granule cells concerning the sources of carbon fuelling the citric acid cycle and the carbon fluxes on different pathways.
Replacement of Ser657 of Protein Kinase C‐α by Alanine Leads to Premature Down Regulation After Phorbol‐Ester‐Induced Translocation to the Membrane
Authors: Gysin Stephan , Imber Roland
Published: July 2004

Protein kinase C (PKC) is activated at the cell membrane by interacting with both the acidic lipid phosphatidylserine and the second messenger diacylglycerol. A direct activation of the kinase is also possible by substituting diacylglycerol with phorbol esters such as the tumor promoting agent 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Transphosphorylation of the activation loop followed by autophosphorylation at sites located on various domains of the protein have been suggested to be required as permissive activation of the α and β isoforms of PKC [Cazaubon, S., Bornancin, F. & Parker, P. (1994) Biochem. J. 301, 443–448; Keranen, L. M., Dutil, E. M. & Newton, A. C. (1995) Curr. Biol. 5, 1393–1403]. Ser657, located near the C‐terminus of PKC‐α, represents a site which is very conserved among the members of the PKC protein family. Circumstantial evidence suggested that this residue represents a possible site of phosphorylation. The conversion of Ser657 to alanine caused a 70% loss of the catalytic activity as well as a drastically increased down regulation upon translocation of this isozyme to the membrane when induced by phorbol ester. The faster electrophoretic mobility of the mutant protein compared to that of the wild‐type enzyme suggested that Ser657 represents a phosphorylation site.
Receptor‐Induced Translocation of Activated Guanine‐Nucleotide‐Binding Protein αi Subunits to the Cytoskeleton in Myeloid Differentiated Human Leukemia (HL‐60) Cells
Authors: Wieland Thomas , Heringdorf Dagmar Meyer Zu , Schulze Rüdiger A. , Kaldenberg‐Stasch Sylvia , Jakobs Karl H.
Published: July 2004

The regulation of the cytoskeletal localization of guanine‐nucleotide‐binding protein αi subunits by formyl peptide receptors was studied in myeloid differentiated human leukemia (HL‐60) cells. Stimulation of formyl peptide receptors with N ‐formyl‐Met‐Leu‐Phe (fMet‐Leu‐Phe) transiently increased the amount of αi subunits in the Triton X‐100‐insoluble cytoskeleton. Similar to the biphasic regulation of the actin content, fMet‐Leu‐Phe (≥10 nM) rapidly increased the cytoskeletal αi content (about threefold at 30 s), which was followed by a rapid reversal to control levels. The formyl peptide receptor increased the cytoskeletal content of both αi subtypes, αi2 and αi3, present in HL‐60 cells. In cells permeabilized with Staphylococcus aureusα‐toxin, fMet‐Leu‐Phe increased binding of the stable GTP analogue, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), to cytoskeletal proteins in a pertussis‐toxin‐sensitive manner, which was completely abolished by the F‐actin‐disrupting agent, cytochalasin B. Using the photoreactive GTP analogue, m‐acetylanilido‐GTP, the formyl peptide receptor‐regulated GTP binding sites at the cytoskeleton were identified as 40‐kDa proteins, the molecular size of αi subunits. Cytoskeleton prepared from stimulated cells did not exhibit increased GTP[S] binding, which suggests that activated αi subunits are translocated to the cytoskeleton. Finally, in α‐toxin‐permeabilized HL‐60 cells, fMet‐Leu‐Phe and GTP[S] cooperatively stimulated actin polymerization. In conclusion, evidence is provided that chemoattractant receptors cause translocation of activated αi subunits to the cytoskeleton coincidentally with F‐actin formation. The data therefore argue for a potential role of translocated αi subunits in the process of receptor‐induced actin polymerization.
The Lipase from Staphylococcus aureus
Authors: Simons Jan‐Willem F. A. , Adams Hendrik , Cox Ruud C. , Dekker Niek , Götz Friedrich , Slotboom Arend J. , Verheij Huhertus M.
Published: July 2004

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N‐terminal hexa‐histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal‐affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short‐chain triacylglycerols and acyl esters of both p ‐nitrophenol and umbelliferone are readily degraded, whereas medium‐ and long‐chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.
A Synthetic Peptide from the Human Immunodeficiency Virus Type‐1 Integrase Exhibits Coiled‐Coil Properties and Interferes with the in Vitro Integration Activity of The Enzyme
Authors: Sourgen Frédéric , Maroun Richard G. , Frère Valérie , Bouziane Mohammed , Auclair Christian , Troalen Frédéric , Fermandjian Serge
Published: July 2004

Integration of the human immunodeficiency virus (HIV‐1) DNA into the host genome is catalysed by a virus‐encoded protein integrase. Here, we report some of the structural and functional properties of two synthetic peptides: integrase‐(147–175)‐peptide reproducing the residues 147–175 (SQGVVESMNKELK159KIIGQVRDQAEHLKTAY) of the HIV‐1 integrase, and [Pro159] integrase‐(147–175)‐peptide where the lysine 159 is substituted for a proline. Circular dichroism revealed that both peptides are mostly under unordered conformation in aqueous solution, contrasting with the α‐helix exhibited by residues 147–175 in the protein crystal structure. In a weak α‐helix‐promoting environment, integrase‐(147–175)‐peptide self‐associated into stable coiled‐coil oligomers, while [Pro159] integrase‐(147–175)‐peptide did not. This property was further confirmed by cross‐linking experiments. In our in vitro experiments, only integrase‐(147–175)‐peptide was able to reduce the integration activity of the enzyme. We propose that the inhibitory activity shown by integrase‐(147–175)‐peptide is dependent on its ability to bind to its counterpart in integrase through a peptide‐protein coiled‐coil structure disturbing the catalytic properties of the enzyme.
Purification and Spectroscopic Characterization of a Recombinant Chloroplastic Hemoglobin from the Green Unicellular Alga Chlamydomonas eugametos
Authors: Couture Manon , Guertin Michel
Published: July 2004

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light‐inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01 % of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non‐covalently bound iron‐protoporphyrin IX heme. The oxy form of the recombinant Hb, purified directly from bacterial cells, is very stable, with a measured half‐life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the LI637 Hb can be reduced by the electron‐transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this, we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low‐spin at physiological pH. These results confirm the existence of a reversible oxygen‐binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.
Temporins, Antimicrobial Peptides from the European Red Frog Rana temporaria
Authors: Simmaco Maurizio , Mignogna Giuseppina , Canofeni Silvia , Miele Rossella , Mangoni Maria Luisa , Barra Donatella
Published: July 2004

A cDNA library from the skin of Rana temporaria has been screened using a cDNA fragment probe that encodes the signal peptide of the precursor of esculentin from the skin secretion of Rana esculentu. With this approach, the cDNAs encoding the precursors of three peptides were isolated. Subsequently, the peptides predicted from the sequence of the cloned cDNAs as well as several structurally related peptides could be isolated from the skin secretion of R. temporuria. These peptides, which were named temporins, have a length of 10–13 residues and show some sequence similarity to hemolytic peptides isolated from Vespu venom [Argiolas, A. & Pisano, J. J. (1984) J. Biol. Chem. 259, 10106–10111]. Natural and synthetic temporins have antibacterial activity against gram‐positive bacteria, but they are not hemolytic. Temporins are the smallest antibacterial peptides hitherto found in nature.
Identification and Characterization of three Isotypes of Protein Phosphatase Inhibitor‐2 and their Expression Profiles during Testis Maturation in Rats
Authors: Osawa Yutaka , Nakagama Hitoshi , Shima Hiroshi , Sugimura Takashi , Nagao Minako
Published: July 2004

cDNAs for three isotypes of inhibitor‐2 (I‐2),I‐2α1, I‐2α2andI‐2βwere isolated from a rat testis library.I‐2α2andI‐2βare new forms. The former is an alternatively spliced form ofI‐2α1, encoding a protein with substitution of three amino acids for 14 amino acids of the I‐2αl protein at the C‐terminus. The latter is derived from a different gene and encodes a 126‐amino‐acid protein having highly conserved regions with the I‐2αl protein from amino acid positions 22–47 and 111–126.I‐2α2andI‐2βare expressed exclusively in the testis, and the expressions of all three forms of I‐2 coincide with sperm cell maturation. The half‐maximal inhibitory concentration of the GST–I‐2α2 fusion protein on the PP1γ2 catalytic subunit is the same as that of GST–I‐2α1, being 10 nM. However, the half‐maximal inhibitory concentration of GST–I‐2β is 100‐fold higher, being 1 μM. GST–I‐2β showed no competition with GST–I‐2α1, and its biological significance is unknown.
Transbilayer Phosphatidylethanolamine Movements in the Yeast Plasma Membrane
Authors: Balasubramanian Krishnakumar , Gupta Chhitar M.
Published: July 2004

Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP‐dependent, protein‐mediated process. To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharomyces cerevisiae (A184D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes. The levels of external PtdEtn in the intact cells were reduced to about 50% by pretreatment of the cells with inhibitors of mitochondrial ATP synthesis, ATPase inhibitors or protein‐sulfhydryl‐group‐modifying reagents, or by depletion of the cells of ATP by metabolic starvation. The levels of external PtdEtn could be restored to normal by repletion of the energy‐depleted cells with ATP. Furthermore, treatment of the energy‐depleted cells with sulfhydryl‐modyfying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells. These results demonstrate an involvement of an ATP‐dependent, protein‐mediated process(es) in the regulation of the PtdEtn distribution across the plasma‐membrane bilayer of yeast cells. The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane.
Structure/Function Relationships in Human Phenylalanine Hydroxylase
Authors: Knappskog Per M. , Flatmark Torgeir , Aarden Johanna M. , Haavik Jan , Martínez Aurora
Published: July 2004

Amino‐terminal and carboxy‐terminal deletion mutagenesis have been used to identify structurally and functionally critical regions of recombinant wild‐type human phenylalanine hydroxylase (wt‐hPAH; Ser2‐Lys452). The wild‐type form consisted of dimeric and tetrameric forms in equilibrium, and only the isolated tetrameric form showed positive cooperativity of substrate (L‐Phe) binding (Hill coefficient h= 2.2, S0.5= 154 μM). The deletion mutants lacking the carboxy‐terminal 24 amino acids hPAH(Ser2‐Gln428) and hPAH(Gly103‐Gln428) formed catalytically active dimers, and incubation with L‐Phe did not promote the formation of tetramers, a characteristic property of dimeric wt‐hPAH. The carboxy‐terminus thus seems to contain a motif required for dimer‐dimer interaction in wt‐hPAH. The deletion mutants hPAH(Asp112‐Lys452), hPAH(Ser2‐Gln428) and hPAH(Glyl03‐Gln428) were all activated by prior incubation with L‐Phe, but did not reveal any positive cooperativity of substrate binding (h= 1.0). The activation by L‐Phe was accompanied by a measurable conformational change (as probed by intrinsic fluorescence spectroscopy) only in the enzyme forms containing the amino‐terminal sequence, i.e. wt‐hPAH and the Ser2 ‐ Gln428 mutant. The amino‐terminal deletion mutants hPAH(Asp112–Lys452) and hPAH(Gly103‐Gln428) revealed high specific activity, increased apparent affinity for L‐Phe (S0.5= 60 μM) and a tryptophan fluorescence emission spectrum similar to that of the L‐Phe‐activated wt‐hPAH. Moreover, prior incubation of the enzyme forms with lysophosphatidylcholine, a commonly used activator of the PAH, only increased the activity of those forms containing the wt‐hPAH amino‐terminal sequence. Our results are compatible with a model in which incubation of wt‐hPAH with L‐Phe induces both a conformational change (with cooperativity in the tetrameric enzyme) which relieves the inhibition imposed by the amino‐terminal domain to the high‐affinity binding of L‐Phe, and an additional activation, as observed for the truncated forms lacking the amino‐terminal.
Role of Zinc‐Finger Proteins Sp1 and Zif268/egr‐1 in Transcriptional Regulation of the Human Synaptobrevin II Gene
Authors: Petersohn Dirk , Thiel Gerald
Published: July 2004

Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5′‐flanking region of the human synaptobrevin II gene is very (G+C)‐rich and contains a 13‐bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr‐1. To test whether Sp1 and zif268/egr‐1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)‐rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr‐1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)‐rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO‐K1 cells together with an expression vector encoding zif268/egr‐1. Zif268/egr‐1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)‐rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)‐rich sequence in Drosophila SL2 cells, which provided an Sp1‐deficient background. Furthermore, a glutathione S ‐transferase fusion protein containing the DNA‐binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter–CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr‐1 ‐binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1‐binding site in this (G+C)‐rich region clearly mediates constitutive transcriptional activation.
Glucocorticoid Receptor Lacking the τ1 Transactivation Domain is a Gene‐Specific Regulator of the Wild‐Type Glucocorticoid‐Receptor Activity
Authors: Delaunay Franck , Liden Johan , Gustafsson Jan‐Åke , Okret Sam
Published: July 2004

The glucocorticoid receptor (GR) contains a major transactivation function (τ1), located in the N‐terminal domain. τ1 contributes to about 80% of the ligand‐inducible transcriptional activity of GR. In this study, we show that GR devoid of τl (AGR) can inhibit activation of gene expression by wild‐type GR but this does not occur for all target genes. Activation of the mouse mammary tumor virus promoter by wild‐type GR in transiently transfected Chinese hamster ovary (CHO) cells lacking endogenous GR was repressed by cotransfecting AGR. This effect was proportional to the amount of transfected ?R and was not due to squelching. A moderate expression level of stably transfected AGR mutant was also shown to repress the transcriptional activity of endogenous GR present in rat skeletal myoblast L8 cells. Glucocorticoid mediated down regulation of endogenous GR gene expression can be blocked by the AGR mutant in stably transfected L8 cells. In contrast, no inhibition was observed on glucocorticoid induction of the endogenous glutamine synthetase gene in L8 cells. However, glucocorticoid induction of a reporter gene driven by the chicken glutamine synthetase promoter was inhibited by ?GR in L8 cells. Stable expression of wild‐type GR in CHO cells rendered the cells glucocorticoid responsive with regard to glutamine synthetase induction but coexpression of ?GR did not repress induction of the endogenous glutamine synthetase gene expression by wild‐type GR. Expression of ?GR alone in CHO cells did not render the glutamine synthetase gene glucocorticoid responsive, indicating that ?GR has no transcriptional activity on the glutamine synthetase gene. We conclude from these results that the structure of glucocorticoid‐response elements within target genes may be very critical for the ability of the mutant receptor to exhibit a dominant negative effect.
Epitope Mapping and Immunoneutralization of Recombinant Human Stem‐Cell Factor
Authors: Mendiaz Elizabeth A. , Chang David G. , Boone Thomas C. , Grant James R. , Wypych Jette , Aguero Bibiana , Egrie Joan C. , Langley Keith E.
Published: July 2004

The epitope regions of three anti‐[stem‐cell factor (SCF)] Ig have been mapped by characterization of immunoreactivities against truncated forms of SCF in immunoblots and against synthetic peptides in solution‐phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli ‐derived human SCF‐(1–164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3–31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61–95 and 95–110, respectively. The epitope of pAb 1337 has been mapped to residues 21–31. The ability of the anti‐SCF Ig to recognize E. coli‐derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied. mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured. mAb 7H6 was found to neutralize in vitro SCF‐mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61–65 and 91–95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF.
Sodium Ion Translocation by N5‐Methyltetrahydromethanopterin: Coenzyme M Methyltransferase from Methanosarcina mazei Gö1 Reconstituted in Ether Lipid Liposomes
Authors: Lienard Tanja , Becher Burkhard , Marschall Mechthild , Bowien Susanne , Gottschalk Gerhard
Published: July 2004

The N5‐methyltetrahydomethanopterin (H4MPT): coenzyme M methyltransferase is a membrane associated, corrinoid‐containing protein that uses the methylation of coenzyme M (HS‐CoM) by methyl‐tetrahydromethanopterin to drive an energy‐conserving sodium ion pump. The enzyme was purified from acetate‐grown Methanosarcina mazei Göl by a two‐step solubilization with η‐octyl‐β‐glucoside, chroma‐tography on hydroxyapatite, and by gel filtration on Superdex 200 or Sepharose CL‐6B. The highly purified protein was apparently composed of six different subunits of 34, 28, 20, 13, 12, and 9 kDa. The N‐terminal amino acid sequences of these polypeptides were determined. The native enzyme exhibited an apparent molecular mass of about 380 kDa. During purification, the enzyme was stabilized with 10 μM hydroxocobalamin. The highest specific activity reached during purification was 10.4 U/mg. The purified enzyme was reconstituted in monolayer liposomes prepared from ether lipids of M. mazei Göl. In experiments with radioactive sodium ions, it was shown that the methyltransferase catalyzes the vectorial translocation of sodium ions across the membrane. Methyltransferase activity was stimulated by sodium ions. 1.7 mol Na+/mol methyl groups transferred were translocated. Methyltetrahydrofolate and methyl‐cobalamin could substitute for methyl‐H4MPT.