bluuebunny/crossref_metadata_2025 huggingface dataset:
Complexes of the polyamines spermine, spermidine and putrescine with α‐lactalbumins
Authors: MOROZOVA Ludmilla , DESMET Johan , JONIAU Marcel
Published: March 2005

The effects of polyamines on the spectral properties and thermal stability of different α‐lactalbumins were measured. Addition of millimolar concentrations of spermine to the Ca2+‐free (apo) form of bovine or goat α‐lactalbumin resulted in spectral shifts, in both the far‐ and near‐ultraviolet ranges, similar to those induced by Ca2+ binding. Fluorescence emission spectra of tryptophan residues underwent a pronounced blue shift, concomitant with a decrease in quantum yield. Also, in the presence of spermine, thermal stability was increased. In contrast, in the case of human and to some extent also equine α‐lactalbumin, the shifts of the CD and fluorescence spectra and the thermal denaturation curves were in the opposite direction.

Direct competition of spermine with Mn2+ could be observed for binding to the Ca2+ site of bovine α‐lactalbumin. As the main binding mode of spermine showed a 1:1 stoichiometry [K1= (2±0.5)×104M−1], we have assumed that binding occurs primarily at this site with one of the distal ammonium groups. In order to accommodate the remaining positively charged ammonium groups of spermine, a binding model was constructed by computer modeling, based on the atomic coordinates of both interacting species. This model makes use of the cluster of negatively charged glutamate residues present in the N‐terminal sequence of bovine α‐lactalbumin, sterically close to the Ca2+ site. The spermine molecule could be nicely fitted in a space delineated by the Ca2+ site, on the one hand, and Glu1, Glu7 and Glu11, on the other. Our model, which has been extended to the shorter polyamines spermidine and putrescine, is able to fully account for the observed stabilization of bovine (and goat) α‐lactalbumins against thermal denaturation, and for the observed CD and fluorescence spectral shifts. It also explains why human (and equine) α‐lactalbumin behave differently.
Human aldehyde dehydrogenase
Authors: KURYS Gloria , SHAH Pritesh C. , KIKONYOGO Alexandra , REED Donna , AMBROZIAK Wojciech , PIETRUSZKO Regina
Published: March 2005

Human liver aldehyde dehydrogenase (E3 isozyme), with wide substrate specificity and low Km for 4‐aminobutyraldehyde, was only recently characterized [Kurys, G., Ambroziak, W. & Pietruszko, R. (1989) J. Biol. Chem. 264, 4715–4721] and in this study we report on its primary structure. Polyclonal antibodies, specific for the E3 isozyme and three oligonucleotide probes derived from amino acid sequence of the E3 protein, were used for isolation of the first cDNA clone encoding the human enzyme (1503 bp; coding for 440 amino acid residues). Additional clones were obtained by using the first isolated clone as a probe. The largest clone of 1635 bp coded for 462 amino acid residues; it was longer at the 3′end of the cDNA non‐coding region. The identity of the clone was established by DNA sequencing and by comparison with peptide sequences derived from the E3 protein, which constituted approximately 29% of the total primary structure of the E3 isozyme. The start codon was never encountered despite a variety of different approaches (500 amino acid residues were expected on the basis of SDS‐gel molecular‐mass determination of the E3 isozyme subunit). Despite the great catalytic similarity between the E3 and E1 isozymes [Ambroziak, W. & Pietruszko, R. (1991) J. Biol. Chem. 266, 13 011–13 018], the primary structure of the E3 isozyme has only approximately 40.6% of positional identity with that of the E1 isozyme. Sequence comparison with GenBank and Protein Identification Resource database sequences indicated no primary structure of aldehyde dehydrogenase more closely resembling the E3 isozyme than that of Escherichia coli betaine aldehyde dehydrogenase (52.7% positional identity), a prokaryotic enzyme specific for betaine aldehyde.
Localization of the cross‐linking site of GPRVVERHK in the γ‐chain of human fibrinogen
Authors: CIERNIEWSKI Czeslaw S. , BUDZYNSKI Andrei Z.
Published: March 2005

The peptide α17–24‐Lys (GPRVVERHK) corresponding to the N‐terminus of the α chain of fibrin was synthesized and used to localize its binding site in the fibrinogen molecule. The peptide was radioiodinated, incubated with fibrinogen, cross‐linked with a bifunctional reagent disuccinimidyl suberate and the resulting product was analyzed in several ways, including plasmin digestion. The binding of the radioactive peptide was mainly to the γ‐chain and was inhibited by unlabelled GPRVVERHK and GPRP. After plasmin digestion, the radioactivity was present in fragment D1 and also in its γ‐chain remnant, but not in fragments D2, D3 or E3. Fragment D1 cross‐linked with iodinated GPRVVERHK was purified by affinity chromatography on immobilized anti‐fragment D IgG, further digested with plasmin in the presence of EGTA and the peptides were fractionated by reverse‐phase HPLC. The amino acid sequence analysis of the radioactive peak revealed the presence of two peptides, γ357–373 and GPRVVERHK. It was concluded that the binding site for GPRVVERHK is in the sequence γ357–373 which is present in fragment D1 but absent in fragments D2 and D3.
Erythritolteichoic acid in the cell wall of Glycomyces tenuis VKM Ac‐1250
Authors: POTEKHINA Natalia V. , TUL'SKAYA Elena M. , NAUMOVA Irina B. , SHASHKOV Alexander S. , EVTUSHENKO Ludmila I.
Published: March 2005

The cell wall of Glycomyces tenuis VKM Ac‐1250 contains the teichoic acid unusual for bacteria. The teichoic acid is a 1,4‐poly(erythritol phosphate) chain composed of about 23 erythritol phosphate units, four or five of which have a 2‐acetamido‐2‐deoxy‐α‐D‐glucopyranosyl substituent at C2(3) of erythritol. The polymer contains O‐acetyl groups. Its structure was identified by chemical analysis as well as 1H‐NMR and 13C‐NMR spectroscopy. No teichoic acid containing a poly(erythritol phosphate) chain has yet been found in Gram‐positive bacteria.
Regulation of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Authors: WENZEL Thibaut J. , LUTTIK Marijke A. H. , VAN DEN BERG Johan A. , STEENSMA H. Yde
Published: March 2005

Expression of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex (PDH complex) and activity of the complex were investigated in cells grown under several conditions. Comparable amounts of PDA1 mRNA and E1α subunit were detected in cells from batch and chemostat cultures grown on various carbon sources, showing constitutive expression of PDA1 at the transcriptional and translational levels. Induction of the regulatory GCN4 mechanism upon histidine starvation, using the anti‐metabolite 3‐amino‐1,2,4‐triazole, increased the levels of PDA1 mRNA by approximately 40%. However, a corresponding increase of E1α concentration or activity of the PDH complex could not be detected. Hence, expression of the PDA1 gene is only regulated to a small extent, if at all, by the GCN4 mechanism.

Contrary to the constant levels of PDA1 mRNA and E1α subunit in both batch and chemostat cultures, the specific activity of the PDH complex varied with the culture conditions. The activity of the PDH complex in chemostat cultures was approximately two–threefold higher than in batch cultures grown on the same carbon sources. Overproduction of the E1α subunit in batch cultures resulted in a two–threefold increase in the activity of the PDH complex. Taken together, these results indicate that the activity of the PDH complex is mainly regulated by post‐translational modification of the E1α subunit.

Expression of PDA1 and activity of the PDH complex were also detected in cultures grown under conditions where no physiological significance of the PDH complex was expected, i.e. during anaerobic growth on glucose or aerobic growth on ethanol. Apparently, the switch from oxidative growth to fermentation occurs without much effect on the PDH complex. These observations suggest that the PDH complex has an alternative function besides sugar catabolism.
Evolution of the monotremes
Authors: RETIEF Jacques D. , WINKFEIN Robert J. , DIXON Gordon H.
Published: March 2005

The protamine P1 genes from two monotremes, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) were isolated after polymerase‐chain‐reaction amplification then cloned and sequenced. The two protamine P1 genes are of 290 bp and 311 bp for platypus and echidna, respectively, and are clearly orthologous to the published sequences of protamine P1 genes of eutherian mammals and birds. Both genes contain an intron, like the mammals and marsupials and unlike the bird P1 genes that are intronless. The deduced protein sequences from the coding areas of the platypus and echidna protamine P1 genes do not contain any cysteine residues. This absence of cysteine residues leaves the sperm nuclei susceptible to disruption in vitro by exposure to increasing ionic strength and is a characteristic of fish, birds and marsupials. In contrast, the P1 protamines of placental mammals invariably contain 6–9 cysteine residues that, as a result of the formation of intermolecular and intramolecular disulfide bridges, significantly increase the stability of the sperm nuclei that can only be disrupted following disulfide‐bond cleavage. Phylogenetic analysis of the protamine P1 gene sequences indicates that the monotremes occupy a position halfway between the eutherian mammals and birds. From the DNA sequences we estimate the time of divergence of the platypus and the echidna to be around 22 million years ago. This data agrees very well with the published estimates of divergence based on other criteria.
Coupling of recombinant 5‐lipoxygenase and leukotriene A4 hydrolase activities and transcellular metabolism of leukotriene A4 in Sf9 insect cells
Authors: MANCINI Joseph A. , EVANS Jilly F.
Published: March 2005

High‐level expression of human leukotriene (LT) A4 hydrolase has been established in Sf9 insect cells using the recombinant baculovirus system. LTA4 hydrolase activity in this system is at least 50‐fold higher than previously achieved in a bacterial cell system. Recombinant viral human LTA4 hydrolase (rvHLTA4h) was used for coinfection studies with recombinant viral 5‐lipoxygenase (rvH5LO). When Sf9 cells expressing 5‐lipoxygenase are incubated in the presence of A23187 and arachidonic acid, (5S)‐hydroperoxy‐6‐trans‐8,11,14‐cis‐eicosatetraenoic acid 5‐H(P)ETE and LTA4 are synthesized in a ratio of 5:1 for 5‐H(P)ETE/LT. Coexpression of 5‐lipoxygenase and LTA4 hydrolase in these insect cells results in the synthesis of 5‐H(P)ETE, LTA4 and in addition LTB4, and the ratio shifts to 2:1 for 5‐H(P)ETE/LT. The production of enzymically formed LTB4 after addition of arachidonic acid to the Sf9 cells coinfected with LTA4 hydrolase and 5‐lipoxygenase is the first demonstration of channeling of arachidonic acid to LTB4 in an engineered intact cell system. This delineates a novel biological system to synthesize significant amounts of the potent chemotactic agent, LTB4. Studies in which Sf9 cells infected with rvH5LO were incubated with Sf9 cells infected with rvHLTA4h resulted in export of LTA4 from the rvH5LO cells and transcellular metabolism of LTA4 to LTB4 in the rvHLTA4h Sf9 cells.
Alanine scanning mutagenesis of human erythropoietin identifies four amino acids which are critical for biological activity
Authors: GRODBERG Jennifer , DAVIS Kerry L. , SYTKOWSKI Arthur J.
Published: March 2005

Erythropoietin regulates the growth and proliferation of red blood cell progenitors. We demonstrated previously the important structural and functional roles of the amino acid region 99–110 in the biological activity of the hormone. [Chern, Y., Chung, T. & Sytkowski, A. J. (1991) Eur. J. Biochem. 202, 225–229]. We have now performed alanine scanning mutagenesis to identify which specific residues in this region are essential for function. Four substitutions reduced the biological activity of erythropoietin. Alanine substitution of Ser104, Leu105 and Leu108 reduced activity to 16, 44, and 37% that of the wild‐type protein, respectively. Most significantly, substitution of Arg103 with Ala reduced activity to undetectable levels, indicating a >230‐fold reduction in specific activity. Immunochemical analyses using anti‐peptide monoclonal antibodies demonstrated that this substitution does not cause major changes in the conformation of the protein or large, localized distortions within the amino acid region 99–129 of the molecule. Heat‐denaturation kinetics for the arginine mutant and wild‐type erythropoietin are virtually identical, further indicating the structural similarity between these two molecules. Based upon these findings, we propose that Arg103 plays an essential role in the biological activity of erythropoietin, presumably by interacting directly with the erythropoietin receptor.
Kinetics of endoglycoceramidase action toward cell‐surface glycosphingolipids of erythrocytes
Authors: ITO Makoto , IKEGAMI Yuko , YAMAGATA Tatuya
Published: March 2005

As shown in the preceding paper [Ito, M., Ikegami, Y., Tai, T. & Yamagata, T. (1993) Eur. J. Biochem. 218, 637–643], endoglycoceramidase (EGCase; EC.3.2.1.123) was found to hydrolyze the cell‐surface glycosphingolipids (GSLs) of erythrocytes without any damage to other cell membrane components. This paper represents the kinetics of EGCase action toward cell‐surface GSLs of erythrocytes. Without activator or detergents, cell‐surface GSLs were found to be hydrolyzed by EGCase II very slowly at pH 7.0. The initial reaction velocity of EGCase II under this condition was 0.038 pmol · min−1· mU−1 for horse erythrocyte cell‐surface GM3 and 0.032 pmol · min−1· mU−1 for guinea pig erythrocyte cell‐surface Gg3Cer. The addition of activator protein (60 μM), which stimulates EGCase II in the absence of detergents, increased the initial reaction velocity of the enzyme 616‐fold for cell‐surface GM3 and 468‐fold for Gg3Cer, while no increased hemolysis was observed with the addition of activator. However, even in the presence of the activator, the cell‐surface GSLs were very resistant to hydrolysis by EGCase II compared to GSL vesicles (or micelles) under same conditions. In contrast to the activator, Triton X‐100 (0.4%, mass/vol.) not only stimulated the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme reaction velocity. The apparent Km and Vmax values of EGCase II were calculated from the Lineweaver‐Burk plot as 47 μM and 35 pmol min−1 mU−1 for horse erythrocyte cell‐surface GM3 and 44 μM and 27 pmol · min−1· mU−1 for guinea pig erythrocyte cell‐surface Gg3Cer, at pH 7.0 in the presence of activator at a concentration of 60 μM.
Recombinant iron‐regulatory factor functions as an iron‐responsive‐element‐binding protein, a translational repressor and an aconitase
Authors: GRAY Nicola K. , QUICK Sabine , GOOSSEN Britta , CONSTABLE Anne , HIRLING Harald , KÜHN Lukas C. , HENTZE Matthias W.
Published: March 2005

The translation of ferritin and erythroid 5‐aminolevulinate synthase mRNAs is regulated via a specific high‐affinity interaction between an iron‐responsive element in the 5′ untranslated region of ferritin and erythroid 5‐aminolevulinate synthase mRNAs and a 98‐kDa cytoplasmic protein, the iron‐regulatory factor. Iron‐regulatory factor was expressed in vaccinia‐virus‐infected HeLa cells (hIRFvac) and in Escherichia coli (hIRFeco). An N‐terminal histidine tag allowed a rapid one‐step purification of large quantities of soluble recombinant protein. Both hIRFvac and hIRFeco bound specifically to iron‐responsive elements and were immunoprecipitated by iron‐regulatory‐factor antibodies. Using in‐vitro‐transcribed chloramphenicol‐acetyltransferase mRNAs bearing an iron‐responsive element in the 5′ untranslated region, specific repression of chloramphenicol‐acetyltransferase translation by hIRFvac and hIRFeco was demonstrated in wheat‐germ extract. In addition, hIRFvac and hIRFeco were shown to display aconitase activity. Treatment of hIRFvac and hIRFeco with FeSO4 resulted in a drastic reduction in iron‐responsive‐element‐binding of iron‐regulatory factor, but caused a strong stimulation of its aconitase activity. The results establish that recombinant iron‐regulatory factor is a bifunctional protein; after purification, it binds to iron‐responsive elements and represses translation in vitro. Following iron treatment, iron‐responsive‐element binding is lost and aconitase activity is gained. No eukaryotic co‐factor seems to be required for the conversion of the iron‐responsive‐element binding to the aconitase form of the protein.
PPQ, a novel protein phosphatase containing a Ser+Asn‐rich amino‐terminal domain, is involved in the regulation of protein synthesis
Authors: CHEN Mao Xiang , CHEN Yu Hua , COHEN Patricia T. W.
Published: March 2005

The sequence of a Saccharomyces cerevisiae cDNA encoding a novel 61‐kDa protein serine/threonine phosphatase, termed PPQ1, is presented. The protein consists of two distinct domains: the carboxy‐terminal phosphatase domain is approximately 60% identical to either PP1 or the carboxy‐terminal domains of PPZ1 and PPZ2, while the amino‐terminal region is rich in serine and asparagine. Deletion of the gene encoding PPQ1 reduces cell growth on several carbon sources, and lowers cell density in the stationary phase. Cells in which PPQ1 gene has been deleted show altered morphology from wild‐type cells in the stationary phase in the absence of an essential amino acid and a reduced rate of protein synthesis in the exponential phase. They are hypersensitive to the protein synthesis inhibitors, cycloheximide and G418, implicating PPQ1 in the regulation of translation.
The biochemistry of white and brown adipocytes analysed from a selection of proteins
Authors: RICQUIER Daniel , CASSARD‐DOULCIER Anne‐Marie
Published: March 2005

CONTENTS –The adipocyte P2/adipocyte lipid‐binding protein–Structure and function – The aP2/ALBP gene–The CCAAT/enhancer‐binding protein–Structure and function – C/EBP genes–Adipsin–The adipsin protein – The adipsin gene – Adipsin and obesity–Lipoprotein lipase–Control of lipoprotein lipase by endocrine factors – The LPL gene–The uncoupling protein–Structure and function – Regulation and control of the UCP gene–The β3‐adrenoceptor–Structure and function – The β3‐AR gene–The type‐II 5′‐deiodinase–Noradrenaline induction of 5′‐DII in brown adipose tissue
A polymerising Root‐effect fish hemoglobin with high subunit heterogeneity
Authors: FAGO Angela , ROMANO Mario , TAMBURRINI Maurizio , COLETTA Massimo , D'AVINNO Rossana , DI PRISCO Guido
Published: March 2005

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the α and β chain has been established. The presence of a reactive Cys in the external position βCD8(49) causes polymerisation through intermolecular disulfide bridges between β chains, with no alteration of functional features.

C. kumu Root‐effect hemoglobin displays very low or no subunit co‐operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH‐dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co‐operativity. In contrast, kinetic heterogeneity of the two chains, which contributes to the observed reduction of co‐operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pKa of the proton‐linked effect on the slow‐reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by Ile. Although this residue has been shown not to significantly alter ligand binding to the α chain, to some extent it can perturb the access of oxygen to the β chain. Thus, this substitution may be the main reason for subunit functional heterogeneity.
Sequence of pig lens aldose reductase and electrospray mass spectrometry of non‐covalent and covalent complexes
Authors: JAQUINOD Michel , POTIER Noelle , KLARSKOV Klaus , REYMANN Jean‐Marc , SOROKINE Odile , KIEFFER Sylvie , BARTH Patrick , ANDRIANTOMANGA Verotiana , BIELLMANN Jean‐François , VAN DORSSELAER Alain
Published: March 2005

The complete sequence of pig lens aldose reductase (EC 1.1.1.21), a member of the nicotinamide coenzyme‐dependent aldo‐keto reductase super family, was determined by the combined use of data obtained from Edman degradation, fast‐atom‐bombardment mass spectrometry and electrospray mass spectrometry. The N‐terminal residue of human and pig aldose reductase was shown to be acetylated. The assignment of a disulfide bridge (Cys298–Cys303) was obtained by mass spectrometry. Electrospray mass spectrometry has been used for molecular mass measurement of human muscle (35758±7Da) and pig lens (35778±3Da) aldose reductase; using mild ionization conditions, it has also been used to study the reversible interaction involved in a non‐covalent complex with NADP+ (36527±4Da). An alkylating analog of NADP+ (3‐chloroacetylpyridine–adenine dinucleotide phosphate) was used as an irreversible inhibitor to investigate the NADP binding site and the mass of the covalent complex was measured (36521±3Da).
Structural and physico‐chemical characteristics of Bordetella pertussis adenylate kinase, a tryptophan‐containing enzyme
Authors: GILLES Anne‐Marie , SISMEIRO Odile , MUNIER Hélène , FABIAN Heinz , MANTSCH Henry H. , SUREWICZ Witold K. , CRAESCU Constantin C. , BARZU Octavian , DANCHIN Antoine
Published: March 2005

The adk gene from the Gram‐negative pathogen Bordetella pertussis was cloned by complementing the thermosensitive Escherichia coli adk strain CR341T28. B. pertussis adenylate kinase is a 218‐amino‐acid protein that has high similarity with adenylate kinase from Escherichia coli and Hemophilus influenzae (57%). A distinct characteristic of enzyme from B. pertussis, not found in other bacterial adenylate kinases, is the presence of a tryptophan residue at position 185. Although distant from the catalytic site, this single tryptophan serves as a convenient probe for monitoring the binding of nucleotide substrates or analogs to the enzyme. Differential scanning calorimetry and equilibrium unfolding experiments in guanidine · HCl indicate similar stabilities for adenylate kinase from B. pertussis and E. coli. An extensive comparison between physico‐chemical properties of adenylate kinase from B. pertussis and the enzyme from E. coli showed that the kinetic and structural properties of the two enzymes are very similar. However, infrared spectroscopy has allowed to identify small but significant differences in the secondary structure of the two proteins.
Reverse micelles as a water‐property‐control system to investigate the hydration/activity relationship of α‐chymotrypsin
Authors: DOROVSKA‐TARAN V. , VEEGER C. , VISSER A. J. W. G.
Published: March 2005

α‐Chymotrypsin, solubilized in hydrated reverse micelles of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in n‐octane, was used as a model system for studying the involvement of different water structures (strongly bound water, disordered water, water clusters and bulk water) in the development of the catalytically active conformation of the enzyme. Results presented in this study indicate a characteristic dependence of the stability/activity profile on the water content of the reverse‐micellar system for values of wo of approximately 5 (wo is defined as [H2O]/[AOT]). The results are consistent with heat‐capacity measurements for proteins. At very low wo values, the conformation of α‐chymotrypsin changes to a very rigid structure in comparison to the structure observed in water. This is demonstrated by the overall center of gravity of the tryptophan fluorescence spectrum of the enzyme at wo= 0.65, which is blue shifted in comparison to the spectrum in bulk water indicating that the enzyme is in an apolar environment. In the absence of a hydration shell, the protein is to a great extent frozen and inactive. A small increase in the level of enzyme hydration (up to wo= 2.3) causes an increase in the amount of strongly bound water associated with the enzyme and the enzyme displays a high catalytic activity. Upon further addition of water, a new unstable water structure with unfavourable enthalpy is developed and the enzyme activity declines, reaching a minimum at wo= 5.1. A new increase of water content within a relatively small range, wo= 5–8, causes a dramatic increase in enzymic activity, reminiscent of a cooperative hydration dependence. In the range wo= 10–29, the effect of hydration on the activity is complete which shows that the enzyme activity depends on the amount of water in contact with the enzyme and not on the total amount of bulk water in the system. The experimental results on enzyme incubation at different wo values followed by dilution to constant high wo, are indicative of inactive conformational substates of α‐chymotrypsin. It is demonstrated that highly active enzyme conformations at very low wo values occur via an induced fit mechanism of substrate binding.
Plasminogen‐activator inhibitor type 2 (PAI‐2) is a spontaneously polymerising SERPIN
Authors: MIKUS Peter , URANO Tetsumei , LILJESTRÖM Peter , NY Tor
Published: March 2005

Plasminogen‐activator inhibitor type 2 (PAI‐2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low‐molecular‐mass form and a secreted, high‐molecular‐mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI‐2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI‐2. With this construct, more than 90% of PAI‐2 was secreted as a glycosylated, 60‐kDa molecular‐mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI‐2, a novel expression vector based on the Semliki‐forest‐virus replicon was used. Secreted PAI‐2 was purified to homogeneity and N‐terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non‐glycosylated form of PAI‐2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI‐2 were found to inhibit plasminogen activators by forming SDS‐resistant complexes and the second‐order rate constants were similar for both forms, ranging over 2.4–2.7×106 M−1s−1 for urokinase‐type PA, 2.5–2.7×105 M−1s−1 for two‐chain tissue‐type PA and 0.8–1.2×104 M−1s−1 for single‐chain tissue‐type PA. None of the purified PAI‐2 forms bound to vitronectin.

Circular‐dichroism spectral analysis revealed that PAI‐2 has a CD spectrum that resembles ovalbumin more than PA‐inhibitor type 1, confirming the greater similarity between these two members of the serine‐protease inhibitor family. Similar to what has been described for the Z‐form of α1‐antitrypsin, purified PAI‐2 was found to spontaneuosly form polymers during incubation at room temperature. Attempts to convert PAI‐2 to a stable locked conformation resembling the conformation of latent PAI‐1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI‐2 polymers, possibly by the loop‐sheet polymerisation mechanism described for α1‐antitrypsin.
Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signalling pathways
Authors: PÜSCHEL G. P. , KIRCHNER C. , SCHRÖDER A. , JUNGERMANN K.
Published: March 2005

Prostaglandin E2 has been reported both to stimulate glycogen‐phosphorylase activity (glycogenolytic effect) and to inhibit the glucagon‐stimulated glycogen‐phosphorylase activity (antiglycogenolytic effect) in rat hepatocytes. It was the purpose of this study to resolve this apparent contradiction and to characterize the signalling pathways and receptor subtypes involved in the opposing prostaglandin E2 actions.

Prostaglandin E2 (10 μM) increased glucose output, glycogen‐phosphorylase activity and inositol trisphosphate formation in hepatocyte cell culture and/or suspension. In the same systems, prostaglandin E2 decreased the glucagon‐stimulated (1 nM) glycogen‐phosphorylase activity and cAMP formation.

The signalling pathway leading to the glycogenolytic effect of PGE2 was interrupted by incubation of the hepatocytes with 4β‐phorbol 12‐myristate 13‐acetate (100 nM) for 10 min, while the antiglycogenolytic effect of prostaglandin E2 was not attenuated.

The signalling pathway leading to the antiglycogenolytic effect of prostaglandin E2 was interrupted by an incubation of cultured hepatocytes with pertussis toxin (100 ng/ml) for 18 h, whereas the glycogenolytic effect of prostaglandin E2 was enhanced.

The EP1/EP3 prostaglandin‐E2‐receptor‐specific prostaglandin E2 analogue Sulproston had a stronger glycogenolytic potency than the EP3 prostaglandin‐E2‐receptor‐specific prostaglandin E2 analogue Misoprostol. The antiglycogenolytic potency of both agonists was equal.

It is concluded that the glycogenolytic and the antiglycogenolytic effects of prostaglandin E2 are mediated via different signalling pathways in hepatocytes possibly involving EP1 and EP3 prostaglandin E2 receptors, respectively.
Evidence for cAMP‐independent thyrotropin effects on astroglial cells
Authors: SAUNIER Bertrand , PIERRE Michel , JACQUEMIN Claude , COURTIN Françoise
Published: March 2005

Thyroid hormones are essential for normal brain development and function. Brain astroglial cells express type II iodothyronine 5′‐deiodinase which converts thyroxine into 3,5,3′‐triiodothyronine. This type II deiodinase is regulated through various signalling pathways, allowing probably for the local adaptation of the level of 3,5,3′‐triiodothyronine. Our results demonstrated that thyrotropin was able to induce type II deiodinase activity in astrocytes. A thyrotropin receptor was demonstrated. It was not coupled, as in thyroid, to adenylyl cyclase and phospholipase C, but it stimulated cytosolic phospholipase A2. The stimulation by thyrotropin of both thyroxine synthesis in thyroid and its local activation in astrocytes, could protect the brain from variations in the level of 3,5,3′‐triiodothyronine.
From anemia to cerebellar dysfunction
Authors: LAMBERT Stephen , BENNETT Vann
Published: March 2005

The focus of this review is on the ankyrin gene family, key elements in the interaction of the spectrin‐based membrane skeleton with the plasma membrane in a variety of tissues and multicellular organisms. The structure/function relationships of ankyrin molecules are reviewed, illustrating how these proteins are uniquely suited to serve as adaptors between the membrane skeleton and a number of integral membrane proteins. Advances in the understanding of ankyrin biology in the brain are discussed and used to show how ankyrins may be involved in the establishment and/or maintenance of specialized plasma membrane domains. Finally, recent research in hematological and neurological disorders are reviewed, suggesting that ankyrins have a role in the development of human disease.
Synthesis and assembly of functionally active human vascular endothelial growth factor homodimers in insect cells
Authors: FIEBICH Bernd L. , JÄGER Birgit , SCHÖLLMANN Claudia , WEINDEL Karin , WILTING Jörg , KOCHS Georg , MARMÉ Dieter , HUG Hubert , WEICH Herbert A.
Published: March 2005

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor with a target‐cell specificity highly restricted to vascular endothelial cells. Recombinant baculovirus were constructed for the production of two different forms of the human VEGF protein in insect cells. VEGF165 and VEGF121 proteins produced by Sf158 cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide‐linked dimer and secretion into the media. Only one of these forms, VEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogenicity by a two‐step heparin‐affinity chromatcgraphy. The biological activity of the purified 43‐kDa homodimer was demonstrated by high‐affinity binding to VEGF receptors, and by the induction of DNA synthesis in vascular endothelial cells. A positive angiogenic activity in vivo was demonstrated by the day‐13 chorioallantoic‐membrane assay. The mitogenic potency of VEGF121 for human umbilical vein endothelial cells was very similar compared to VEGF165. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide‐bridge formation can be efficiently expressed in high concentration (up to 5 μg/ml) in insects cells.
High‐yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli
Authors: POMPEJUS Markus , FRIEDRICH Karlheinz , TEUFEL Michael , FRITZ Hans‐Joachim
Published: March 2005

A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis. The gene sequence was designed for convenient manipulation in future protein engineering experiments. In‐frame fusion of bos to the lacZ454 gene allowed high‐yield production in Escherichia coli of a β‐Gal454/BO fusion protein, deposited as intracellular inclusion bodies. These were enriched by virtue of their insolubility in 0.5% Triton X‐100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the β‐Gal454/BO boundary. Pure BO could be obtained from the mixture of water‐insoluble cleavage products by selective extraction into organic solvent. The yield was in the range 30–50 mg pure protein/1 culture medium, depending on individual preparation. This material could be used for reconstitution of fully functional bacteriorhodopsin. Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.
Snake‐venom phospholipase A2 neurotoxins
Authors: CHOUMET Valérie , SALIOU Bernard , FIDELER Laure , CHEN Yuang‐Cong , GUBENSEK Franc , BON Cassian , DELOT Emmanuèle
Published: March 2005

The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (β‐neurotoxins): single‐chain β‐neurotoxins, such as agkistrodotoxin and ammodytoxin‐A, and dimeric β‐neurotoxins, which, in the case of the best studied ones, crotoxin‐like toxins, consist of the non‐covalent association of a phospholipase A2 (CB) and a non‐enzymatic chaperon (CA). Possible evolutionary relationships of these β‐neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex.

CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti‐CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone.

A 10‐fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA agkistrodotoxin. Ammodytoxin A did not compete with the anti‐CA mAb.

These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).
Enzyme inactivation and protection during entrapment in reversed micelles
Authors: KHMELNITSKY Yuri L. , HILHORST Riet , VISSER Antonie J. W. G. , VEEGER Cees
Published: March 2005

Lactate dehydrogenase (LDH) was found to lose rapidly its catalytic activity during the process of entrapment in hydrated reversed micelles of sodium bis(2‐ethylhexyl)sulfosuccinate (AOT) in octane. It was demonstrated that this inactivation was caused by the surfactant which penetrated into the injected enzyme‐containing aqueous bulk phase during the short time of mechanical stirring needed to convert the initial biphasic mixture into a monophasic reversed micellar solution. The unfavorable inactivation phenomenon could be efficiently eliminated by the addition of either NADH or pyruvate into the enzyme stock solution prior to its injection into AOT solution in octane. The catalytic activity of substrate‐protected LDH in AOT reversed micelles increased with increasing water content of the micellar system, reaching the maximal level above wo= 30 when aqueous inner cores of reversed micelles grew large enough to allow unrestricted accommodation of the enzyme molecule. It is suggested that the employment of substrate‐protected enzymes could represent a generally useful approach for producing highly efficient enzyme‐containing reversed micellar reaction systems.
Rat kidney endopeptidase 24.16
Authors: BARELLI Hélène , VINCENT Jean‐Pierre , CHECLER Frédéric
Published: March 2005

Endopeptidase 24.16 was purified from rat kidney homogenate on the basis of its ability to generate the biologically inactive degradation products neurotensin (1–10) and neurotensin (11–13). On SDS gels of the proteins pooled after the last purification step, the enzyme appeared homogeneous and behaved as a 70‐kDa monomer. The peptidase was not sensitive to specific inhibitors of aminopeptidases, pyroglutamyl aminopeptidase I, endopeptidase 24.11, endopeptidase 24.15, proline endopeptidase and angiotensin‐converting enzyme but was potently inhibited by several metal chelators such as o‐phenanthroline and EDTA and was blocked by divalent cations. The specificity of endopeptidase 24.16 towards peptides of the tachykinin, opioid and neurotensin families was examined by competition experiments of tritiated neurotensin hydrolysis as well as HPLC analysis. These results indicated that endopeptidase 24.16 could discriminate between peptides belonging to the same family. Neurotensin, Lys8‐Asn9‐neurotensin(8–13) and xenopsin were efficiently hydrolysed while neuromedin N and kinetensin underwent little if any proteolysis by the peptidase. Analogously, substance P and dynorphins (1–7) and (1–8) were readily proteolysed by endopeptidase 24.16 while neurokinin A, amphibian tachykinins and leucine or methionine enkephalins totally resisted degradation.

By Triton X‐114 phase separation, 15–20% of endopeptidase 24.16 partitioned in the detergent phase, indicating that renal endopeptidase 24.16 might exist in a genuine membrane‐bound form. The equipotent solubilization of the enzyme by seven detergents of various critical miscellar concentrations confirmed the occurrence of a membrane‐bound counterpart of endopeptidase 24.16. Furthermore, the absence of release elicited by phosphatidylinositol‐specific phospholipase C suggested that the enzyme was not attached by a glycosyl‐phosphatidylinositol anchor in the membrane of renal microvilli. Finally, endopeptidase 24.16 could not be released from these membranes upon trypsinolysis.
Interaction of staphylokinase with different molecular forms of plasminogen
Authors: LIJNEN H. Roger , VAN HOEF Berthe , COLLEN Désiré
Published: March 2005

In order to obtain more information on the mechanism of plasminogen activation by staphylokinase (STA), we have studied the interaction between recombinant STA (STAR) and different molecular forms of human plasminogen, including Glu‐plasminogen (native moiety), Lys‐plasminogen (partially degraded moiety) and low‐molecular‐mass (LMM) plasminogen (moiety lacking kringles 1–4).

Addition of 2 μM STAR to 0.4 μM Glu‐plasminogen, Lys‐plasminogen or LMM plasminogen resulted in the generation of proteolytic activity towards the chromogenic substrate D‐Val‐Leu‐Lys‐NH‐PhNO2 (S‐2251) corresponding to the exposure of 1 active center/plasminogen molecule. Complex formation was associated with conversion of the one‐chain plasminogen moieties to two‐chain plasmin, and with quantitative conversion of Glu‐plasminogen to Lys‐plasmin. The stoichiometry of the plasminogen‐STAR complex, determined by binding of the complex to Lys‐Sepharose and measurement of residual STAR, was found to be equimolar. The plasminogen‐STAR complexes were inhibited by α2‐antiplasmin with second‐order rate constants of 2.4 ± 0.17 × 106 M−1 s−1 for Glu‐plasminogen, 2.4 ± 0.21 × 106 M−1 s−1 for Lys‐plasminogen and 9.4 ± 1.5 × 104 M−1 s−1 for LMM plasminogen. Glu‐plasmin‐STAR, Lys‐plasmin‐STAR and LMM plasmin‐STAR had comparable catalytic efficiencies (kcat/Km) for the activation of Glu‐plasminogen (0.24–0.29 μM−1 s−1), Lys‐plasminogen (0.57–0.79 μM−1 s−1) or LMM plasminogen (0.11–0.16 μM−1 s−1).

In a human plasma milieu in vitro STAR, Glu‐plasmin‐STAR, Lys‐plasmin‐STAR and LMM‐plasmin‐STAR were equally effective for the lysis of 125I‐fibrin‐labeled human plasma clots [50% clot lysis in 2 h (EC50) with 11–13 nM test compound] and equally fibrin‐selective (residual fibrinogen levels of 72–84% after 2 h at EC50).

Our results thus confirm that plasminogen and STAR form a 1:1 stoichiometric complex in which plasminogen is converted to plasmin and Glu‐plasminogen to Lys‐plasmin. The lysine‐binding sites in kringles 1–4 of plasminogen are not required for the complex formation with STAR, nor for the enzyme activity of the complex with STAR in purified systems and in a human plasma milieu. The lysine‐binding sites are, however, important for the rate of the inhibition of the complexes by α2‐antiplasmin.
The reaction mechanism of Ca2+‐ATPase of sarcoplasmic reticulum
Authors: LACAPERE Jean‐Jacques , GUILLAIN Florent
Published: March 2005

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5°C, the association and dissociation rate constants of Mg · ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 μM calcium and 5 mM MgCl2 (3–4x 106 M−1· s−1 and 9 s−1 respectively). Therefore, we have determined the true affinity for Mg · ATP (Kd= 3 μM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg · ATP binding is the rate limiting step in the phosphorylation process, and Mg · ATP dissociation is slower than dephosphorylation.

The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two‐step mechanism, both steps being accelerated by Mg · ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca2+ ‐ATPase, we showed that rate constants for calcium binding are always lower than those of Mg · ATP binding to an EGTA‐incubated enzyme. We measured dissociation and association rate constants of Mg · ATP binding in the absence of calcium (k‐1= 25 s−1 and k1= 7.5 106 M−1· s−1). This gives a Kd similar to that obtained by equilibrium measurements (3–4 μM).

Both non‐phosphorylated conformations of the enzyme have similar affinity for Mg · ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non‐phophorylated enzyme for Mg · ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca2+‐ATPase cycle, in which ATP is sequentially substrate and activator on a multiple‐function single site.
Partial cloning of a squid microtubule‐associated protein (MAP H1) and the identification of the microtubule binding domain
Authors: KRONIDOU Nafsika G. , SLOBODA Roger D.
Published: March 2005

An approximately 420‐kDa ATP binding protein, referred to as MAP H1, has previously been shown to be involved in microtubule‐dependent vesicle motility in the squid giant axon. To gain further insight into the structure and function of this protein, partially overlapping cDNA clones encoding approximately a quarter of the MAP H1 molecule were identified from two squid optic lobe libraries using affinity‐purified antibodies to squid MAP H1. One clone in particular (KS18), which hybridizes to an approximately 13‐kb message, encodes a series of almost identical repeats of a 16‐amino‐acid sequence that is tandemly repeated. The sequence of clone KS18 is unique and does not correspond to any nucleotide or amino acid sequence in the data base. The presence of repeated elements within the microtubule binding domain of several other MAPs prompted us to investigate whether the MAP H1 repeats are involved in microtubule binding. In‐vitro‐synthesized polypeptides containing these repeats sediment with taxol‐stabilized microtubules in a microtubule binding assay. The predicted secondary structure of the 16‐amino‐acid repeat region of MAP H1 contains alternating β‐sheets and turns and could form the globular domain seen in negative‐stain electron micrographs of MAP H1.
NADH binding site and catalysis of NADH peroxidase
Authors: STEHLE Thilo , CLAIBORNE Al , SCHULZ Georg E.
Published: March 2005

The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X‐ray data collection, model building of NADH and refinement at 0.24‐nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325–1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and sulfenic acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation‐sensitive sulfenic acid and propose a reaction mechanism.
High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor
Authors: PAPADOULI Irene , SAKARELLOS Constantinos , TZARTOS Socrates J.
Published: March 2005

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti‐MIR monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67 – 76 of the AChR α‐subunit. A thorough study of the epitope(s) for anti‐MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data.

Use of all possible overlapping hexapeptides within Torpedo and human α40 – 91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide α67 – 71. Systematic screening of peptide analogues, where each amino acid residue within α67 – 76 and α67 – 74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti‐MIR mAb binding, whereas Pro69 and Ala/Asp70 were less but still significantly important. mAb binding to α67 – 76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of α76 or combinations of two substitutions at α73 and α76 were tested. Several of these analogues (mainly His76, Arg76, Val73Ala76, His73Ala76, Val73Arg76) exhibited dramatic mAb binding enhancement. Some anti‐MIR mAbs that do not bind to α67 – 76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.